Huntington’s disease (HD) is a disorder caused by an abnormal expansion of trinucleotide CAG repeats within the huntingtin (Htt) gene. Under normal conditions, the CREB Binding Protein interacts with CREB elements and acetylates Lysine 27 of Histone 3 to direct the expression of several genes. However, mutant Htt causes depletion of CBP, which in turn induces altered histone acetylation patterns and transcriptional deregulation. Here, we have studied a differential expression analysis and H3K27ac variation in 4- and 6-week-old R6/2 mice as a model of juvenile HD. The analysis of differential gene expression and acetylation levels were integrated into Gene Regulatory Networks revealing key regulators involved in the altered transcription cascade. Our results show changes in acetylation and gene expression levels that are related to impaired neuronal development, and key regulators clearly defined in 6-week-old mice are proposed to drive the downstream regulatory cascade in HD. Here, we describe the first approach to determine the relationship among epigenetic changes in the early stages of HD. We determined the existence of changes in pre-symptomatic stages of HD as a starting point for early onset indicators of the progression of this disease. Within genetic pathologies, there is a group of diseases that involve non-traditional inheritance mechanisms characterized by highly unstable mutations called expansions . Most ofHD is an autosomal dominant neurodegenerative disorder caused by the expansion of the PolyQ segment equal to or greater than 36 triplet repeats in exon 1 of the Huntingtin gene (Htt), thereby producing a polyglutamine tract in the Huntingtin protein ,5,6,7. HTo date, the precise cellular function of the HTT protein, encoded by the Htt gene, has not been yet elucidated ,20,21, aCBP can also activate gene transcription through its intrinsic histone acetyltransferase activity , adding Huntington’s disease has been extensively studied in symptomatic stages at genomic, epigenomic, and proteomic levels, finding that the lengths of the CAG segment of the Huntingtin gene influence alterations in acetylation levels, which translates into changes in regulatory events and ultimately guides neurodegenerative processes and cellular and glial identity ,37,38. AThe alteration of gene expression in striatal neurons is an early event described in HD that starts before neuronal death, being nuclear and cytoplasmatic mHtt inclusion. However, the gene expression profile of the R6/2 HD model before the striatal neuronal loss is not yet described. We first see the presence of mHtt accumulation in striatum tissue at 6, 9, and 13 weeks in the R6/2 model. We observed a progressive increase in mHtt aggregation levels in the striatum and cortex brain region . These rTo determine the presence of transcriptomic alterations in the early stages of HD, we performed a differential gene expression analysis. To do so, striatal tissue from both Wild-Type (WT) and R6/2 mice models at 4 and 6 weeks were analyzed . We founFor a better understanding of processes in which the DE participate, we performed an enrichment analysis of gene ontology (GO) terms. There was no significant enrichment for DE genes for GO biological processes’ molecular functions at four weeks old. Whereas for six weeks old R6/2 mice, we found enriched GO terms related to cell communication functions that could be associated with neurological events present in the context of HD such as synapse organization, cognition, learning, and locomotory behavior for biological process and channel activity for molecular function . RegardiAs described in other murine models of the disease at symptomatic stages, there exists a variation in the acetylation levels in this disease ,42. ThusWhen looking for changes in the acetylation levels for putative CREB and CBP binding sites , 635 CREWe next contextualized a general GRN considering the quantification of gene expression carried out in this work for the R6/2 murine model at four and six weeks of age. For the two ages, both conditions are similar according to the number of nodes and edges presented in the networks, with few elements presented in only one condition, most of them in the HD model, being associated with the regulation of transcriptional processes in four-week mice see . AdditioLooking for a relationship between differentially acetylated regions and DE genes in 4-week-old mice, we did not find a direct association , but theRegarding the determination of Master Regulators (MRs), in the four week old stage, we did not find that TF belongs to the MR family . MoreoveOn the other hand, 4 and 24 of these MRs are up/down regulated in the HD condition , which cHuntington’s disease is a neuropathology with no cure and whose treatments focus on improving the patient’s quality of life. Current work related to this disease has mainly focused on late stages using the R6/2 murine model ,42, whilIn this work, we have addressed how changes in the acetylation patterns in the chromatin shape the transcriptomic landscape in the pre-symptomatic stages of Huntington’s disease that have not been addressed in-depth to date in a genomic/epigenomic context, focusing on the integration of data associated with gene expression and changes in H3K27 acetylation levels. We aimed to increase current knowledge on the early stages of HD, trying to identify those regulatory processes whose functional impairment may serve as an early indicator of the disease.Our results show that non-symptomatic mice present change in their transcriptomic levels when contrasting the HD model versus the healthy condition. Importantly, this increase in the number of DEG shows a direct relationship, whereas when the mice’s ages increase, the number of DEG increases. Overall, an analysis of the functions and pathways in which these DEGs participate shows an enrichment of functional terms linked to ion transport and learning, terms that are related to the functions performed by the striatum, and which are affected as the disease progresses ,44. At fhttps://www.malacards.org/card/spinal_muscular_atrophy_distal_x_linked_3, accessed on 15 June 2023).Furthermore, it is also striking that three genes, Egr1, Rec8, and Gm12695, are DE in the three analyses; both Egr1 and Rec8 are downregulated in HD, while Gm12695 is overexpressed. Egr1 encodes a TF that participates in transcriptional repression and synaptic plasticity processes , which aRegarding known DEG genes in the HD condition also found in our analysis, Crapser and colleagues found a number of genes at eleven weeks which are downregulated . Among tum Scn4b ,48 and Sum Scn4b . In our um Scn4b .When analyzing these differentially acetylated promoters, we did not find GO term enrichment , but according to our predictions, at 6 weeks, genes that are being regulated by CRE elements act on neurotransmission pathways, which agrees with previous reports that showed a higher neurotransmitter release in the early stages of the disease. One possible cause for this altered release is the dysfunction and death of MSN striatal neurons, which has been related to the origin of motor disorders associated with this disease . By lookThe integration of information about differential acetylation levels in promoters and gene expression levels in the gene regulatory networks shows us that for both time points under study, the transcriptional processes are similar . AdditioFinally, we could not determine master regulators guiding the changes in striatal tissue neurons at four weeks following our approach. This agrees with our findings regarding differential expression and reduced variation in acetylation in the earliest developmental stage we analyzed. However, we have been able to determine several master regulators at six weeks according to the definition proposed by Davis and Rebay , where aThis work consists of a first approach to determine the relationship among epigenetics changes in the HD and how it could be affecting transcriptional levels occurring in the early stages of Huntington’s disease. Therefore, further work could validate the role of genes coding for the MR we found as to be related to early HD response as shown in the network analysis. Moreover, genes shown to be linked to these MR and their functions would be a starting point to drive the discovery and determination of possible therapeutic targets and treatments to attack this disease, given that current diagnosis relies on symptom detection or genetic tests that are usually performed when there is the genetic background to do so. In addition, it is crucial to establish a connection between alterations in other histone modifications and H3K27ac, taking into account previous studies that have reported changes in the levels of H3K4me3, H3K9ac, H3K12ac, and H3K14ac in HD ,72,73. TStriatal tissue samples were obtained from R6/2 female mice at their fourth and sixth week of life. The R6/2 transgenic model that contains exon 1 of the mHtt gene human was obtained from The Jackson Laboratory with all animal protocols abiding by the animal management protocol approved by the Bioethics and Biosafety Committee of the Universidad Mayor.® reagent . The total RNA extraction was digested with DNAse I to avoid contamination of the sample with genomic DNA. The RNA concentration was then measured with the Quant-iT ™ RiboGreen® RNA Assay Kit , and its integrity was determined by the Bioanalyzer 2100 kit . RNA libraries were prepared with the Illumina TruSeq TM RNA Sample Preparation Kit, following the manufacturer’s protocol. The rRNA-depleted RNA was fragmented using divalent cations at elevated temperatures. First-strand cDNA synthesis produced single-strand DNA copies from RNA fragmented by reverse transcription. After second-stranded cDNA synthesis, the double-stranded DNA will be repaired at the 3 ’ends. Finally, PCR ligated the universal adapters to the cDNA fragments to produce the final sequencing library. After library validation was carried out with the DNA 1000 chip , the samples were pooled in equal concentrations into one pool and run on the Illumina HiSeq equipment for 100 base pair-end sequencing.Samples of striatal tissue from 4- and 6-week-old R6/2 mice and respective controls were obtained in triplicate. Total RNA was purified by tissue homogenization using Trizolhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 15 June 2023; Then, adapters were removed with Trimmomatic [The RNA-seq Raw data identified so far for striatal tissue samples of animal models of HD R6/2 for 4 and 6 weeks was analyzed using a pipeline explained as follows: first, the quality of the RNAseq data readings was analyzed through FastQC mmomatic . After tmmomatic , settingmmomatic  over alimmomatic  and usinmmomatic , contrasg, resuspended in 0.3 mL of sonication buffer , and incubated for 10 min on ice extracts from Chromatin sonicated for 15 min, in cycles of 30 s on and 30 s off in BioruptorPico , centrifuged at 16,000× g for 15 min at 4 °C to obtain fragments of 500 bp or smaller. The supernatant was collected, divided into aliquots, frozen in liquid nitrogen, and stored at −80 °C; an aliquot was used for quantification by Qubit fluorometric quantification .ChIP-Seq were performed on cross-linked chromatin samples, with the following modifications. The tissues were washed with a cold 1 × PBS buffer, incubated for 10 min with 1% formaldehyde (FA) at room temperature, and washed again with 1 × PBS. For ChIP against chromatin-modifying enzymes, double crosslinking with EGS (ethylene glycol bis)  was used; FA crosslinked tissue was incubated with EGS for 1 h at room temperature with gentle agitation, and was washed three times with cold PBS, resuspended in 1 mL of cell lysis buffer  and homogenized with a Dounce homogenizer  (25 times using a mortar). The cell extract was collected by centrifugation at 3000× g for 5 min, and the supernatant was collected and immunoprecipitated with specific antibodies for 12–16 h at 4 °C. The immune complexes recovered with an additional 50 g for 5 min. Protein-DNA complexes were eluted by incubation with 100 3 and 1% SDS) for 15 min at 65 °C. Extracts were incubated for 12–16 h at 65 °C, to reverse crosslinking the proteins digested with 100 Chromatin size was confirmed by an electrophoretic analysis . ExtractThe right cerebral hemisphere was dissected into the different areas of the brain to be analyzed. The tissues were immediately transferred to dry ice and stored at −80 °C for further analysis. The brain samples were homogenized in RIPA with 1% SDS and a protease inhibitor cocktail  using a sonicator with a 10 s pulse at 40% amplitude. Protein concentrations were estimated using the BCA assay . Cell lysates was denatured under reducing conditions by boiling 30 g for 20 min. The supernatant was recovered as the ”Soluble“ fraction. The pellet was washed with 500 g for 5 min after each wash. Then, the pellet was resuspended with 150 The brain samples were homogenized in Soluble lysis buffer , douncing slowly 30 times in a 1 mL glass douncer. The homogenate was transferred into labeled, ”Insoluble“ tubes and lyse on ice for 1 h. The samples were centrifuged at 4 °C at 15,000×p-value of 0.05. The acetylation peaks obtained in each sample and those common to both ages under study were analyzed using Bedtools software [p-value <0.05.Sequencing results were analyzed using a bioinformatic pipeline, similar to the transcriptomic analysis, to identify changes and/or similarities in H3K27ac levels in striatal tissue as follows. First, the quality of the reads in the ChIP-seq data was analyzed via FastQC; next, adapters were removed with Trimmomatic ; the Gensoftware  using thsoftware , settingsoftware  and finasoftware , considep-value of <0.05.Differentially acetylated promoters were identified following the same steps to identify H3K27ac levels. However, after alignment of the reads using Bowtie against the mouse genome version GRCm38/mm10, the count of the reads that align against each promoter was computed using Htseq-count , using iCRE-regulated candidate genes were identified using specific distance thresholds from the transcription start site and the occurrence of CREB-binding motif sequences in the genome. To identify CRE-binding regions in the mouse genome, we first downloaded CREB-binding motifs of CRE elements from the JASPAR database  scanned Considering the expressed genes in both models (R6/2 and their controls at 4 and 6 weeks old), in total, four gene regulatory networks were developed as described. Briefly, we kept outgoing edges of expressed TFs from the union of high confidence general gene regulatory networks (GRNs) deposited in TRRUST , RegNetwThe Master Regulators (MRs) search in our contextualized networks was performed as follows. For genes defined as differentially expressed, we select their first and second upstream neighbors in both networks similarly . Then, w


The polyplacophoran mollusks (chitons) possess serially arranged shell plates. This feature is unique among mollusks and believed to be essential to explore the evolution of mollusks as well as their shells. Previous studies revealed several cell populations in the dorsal epithelium (shell field) of polyplacophoran larvae and their roles in the formation of shell plates. Nevertheless, they provide limited molecular information, and shell field morphogenesis remains largely uninvestigated.Acanthochitona rubrolineata based on morphological characteristics and molecular patterns. A total of four types of tissue could be recognized from the shell field of A. rubrolineata. The shell field comprised not only the centrally located, alternatively arranged plate fields and ridges, but also the tissues surrounding them, which were the precursors of the girdle and we termed as the girdle field. The girdle field exhibited a concentric organization composed of two circularly arranged tissues, and spicules were only developed in the outer circle. Dynamic engrailed expression and F-actin (filamentous actin) distributions revealed relatively complicated morphogenesis of the shell field. The repeated units (plate fields and ridges) were gradually established in the shell field, seemingly different from the manners used in the segmentation of Drosophila or vertebrates. The seven repeated ridges also experienced different modes of ontogenesis from each other. In the girdle field, the presumptive spicule-formation cells exhibited different patterns of F-actin aggregations as they differentiate.In the present study, we investigated shell field development in the chiton These results reveal the details concerning the structure of polyplacophoran shell field as well as its morphogenesis. They would contribute to exploring the mechanisms of polyplacophoran shell development and molluscan shell evolution.The online version contains supplementary material available at 10.1186/s13227-023-00209-9. Polyplacophora, whose extant members are called chitons, represents a unique lineage of mollusks. In particular, polyplacophorans possess eight shell plates aligned in a serial pattern along the anteroposterior axis  Fig. 1aa1a. ThisIn development, seven serially arranged units of tissues are formed on the dorsal epithelium of polyplacophoran larvae, which then produce seven shell plates after metamorphosis –11; the engrailed [hox genes [engrailed, which is expressed in cells not involved in shell plate secretion [The polyplacophoran shell fields contain distinct cell populations, with different roles in the formation of shell plates and the connective tissues , 11. On ngrailed , key devox genes –15 and oox genes , 17. Nevecretion . While tecretion , 11, addRhyssoplax olivacea (= Chiton olivaceus) [Given the distinct cell populations in different compositions of the shell field , 11, it ivaceus) , and genivaceus) , 14, 16.Acanthochitona rubrolineata. The results revealed details regarding the molecular patterns of different tissues inside the shell field, as well as those concerning their morphogenesis. These results add to the knowledge of polyplacophoran shell development and provide insights into molluscan shell evolution.The morphogenesis of shell field establishes the framework for subsequent shell plate secretion, and thus investigations of this process would be essential to understand polyplacophoran shell formation. Moreover, given that development often provides important evolutionary implications, investigating the early development of polyplacophoran shell field may also provide clues to explore evolutionary issues, such as the origin of shell plates, the evolutionary relationships among the sclerotized structures of mollusks/spiralians, and even the evolution of animal segmentation , 8, 18. A. rubrolineata is a free spawning species, which directly releases sperm and oocytes into the water column. As in many chitons, a key feature of A. rubrolineata oocyte is that it is enclosed by an elaborate egg hull showing extensive superficial protrusions . The early larvae at 12–16 hpf showed no recognizable dorsal structures Fig. a–c, f–h.The lateral cilia in the posttrochal region could be discerned at 14 hpf Fig. g. Very cSpecification in the central region of the shell field became evident at around 22 hpf, when alternative bulges and grooves were discernable Fig. k. Soon aAs SEM revealed continuous morphogenetic changes during the development of the shell field, we investigated the dynamics of filamentous actin (F-actin) to explore more details. In particular, CLSM  3D projections were carefully prepared to avoid influences from strong staining in larval muscles  became further stronger with ontogenesis Fig. l, m, indengrailed [A. rubrolineata, engrailed expression was detected in both dorsal and ventral tissues. We previously revealed that the dorsal domain of engrailed expression was related to shell development [We finally investigated the expression of the well-known shell-formation gene ngrailed . In A. relopment .engrailed could be detected at 12 and 14 hpf, which was scattered distributed in both pretrochal and posttrochal regions and it was difficult to conclude a clear pattern  [Ischnochiton rissoi (= Ischnochiton rissoa) and Cyanoplax caverna (= Lepidochitona caverna) [engrailed positive cells are revealed in A. rubrolineata  [One notable fact is that the plate fields exhibited significant invagination in lls Fig. d; a simi cinera) . In contcaverna) , 12, indata Fig. o and C. caverna) . Howevercaverna) , 21, theI. rissoi (= I. rissoa), including types 3 and 4 in the plate field producing the shell plates and types 1 and 2 in the intersegmental ridges that generate connective tissues between shell plates [C. caverna (= L. caverna), and type 1 and 2 cells are further demonstrated to express engrailed [Kniprath identified four cell types (1–4) in the central region of the shell field in l plates . Type 1 ngrailed . Our resA. rubrolineata (between 22 and 24 hpf), making it difficult to explore the details of their morphogenesis. We thus traced the dynamics of related molecules to reveal more details. The engrailed expression was continuously changing before the formation of morphologically discriminable ridges (16–24 hpf), suggesting relatively complicated development. The key characteristic of engrailed expression is that it initially showed no clear patterns (12–14 hpf), and gradually transited into striped pattern later (16–24 hpf). This mode of development seems to be different from the formation of striped gene expression of segmentation genes in vertebrates or Drosophila, indicating that the serial ridges (and plate fields) may be formed through different manners from those used in the segmentation of vertebrates or Drosophila . First, different stripes of engrailed expression experienced varied modes of changes. In particular, stripes 1 and 2 formed individually, but other five stripes were derived from two expression areas, each of which transitioned into two (no. 3–4) or three (no. 5–7) stripes later. These different modes of genesis should provide useful clues to explore the underlying regulatory mechanisms. For instance, the formation of anterior and posterior engrailed stripes (ridges) may be regulated by varied mechanisms, reminiscent of the varied regulatory mechanisms underlying the formation of anterior and posterior somites in amphioxus [engrailed generally follow an anterior-to-posterior pattern . It then enhanced with the invagination of plate fields  [The girdle (or perinotum) of adult chiton encircles the shell plates and develops spicules on it. It is proposed that the epithelium of the girdle is not so different from that producing the shell plates . From thl origin –8, we prld Figs.  and 7. Aprearum) , the girengrailed expression. This is consistent with the previous observation that only the larval girdle without engrailed expression develop spicules [The outer girdle field likely contained spicule-formation tissues that exhibited superficial tiny protrusions and F-actin aggregations (“spots” or “tubes” at different stages) but lacked spicules . It was spicules . We founA. rubrolineata is also required.The demonstration of the correlation between characteristic F-actin aggregations and spicule formation would contribute to several evolutionary issues concerning aculiferan spicules. First, as two representatives of extant aculiferan mollusks, the common feature of polyplacophorans and aplacophorans is the ability to develop spicules, and thus spicule development should contain important information of aculiferan evolution. Although almost the same process of spicule formation is suggested in two aculiferan clades , moleculC. caverna (= L. caverna) [engrailed corresponding to the gap between the outer girdle field (marked by F-actin aggregations) and the central region. We interpreted these circularly organized cells to be a part of the girdle field, and termed them as the inner girdle field. Although the roles of these cells are not fully elucidated, it is reported that they are not involved in spicule development and contain some secretory cells [engrailed-expressing tissue in the shell field as a whole, it is attempting to propose that the engrailed expression seems to define the boundary of shell plates: that in the inner girdle field demarcates the entire region bearing all shell plates, and the expression in ridges delineates each plate. A similar idea regarding the role of engrailed in determining developmental boundaries was proposed previously [Consistent with the report in caverna) , we founry cells , 12. Morengrailed in intersegmental ridges and inner girdle field indicates similarities between related cells. This is consistent with the report that type 1 and 2 cells are distributed in both ridges and girdle of I. rissoi (= I. rissoa) [engrailed expression and F-actin aggregations are adjacent to each other but never intermixed.The common expression of  rissoa) . SimilarA. rubrolineata. We found that the tissues right adjacent to the prototroch, in both pretrochal and posttrochal regions, all expressed engrailed  shell plates would develop. The girdle field itself also showed a comparable concentric organization, comprising two circles of F-actin “spots/tubes” (outer girdle field) and engrailed expression (inner girdle field).The shell field represents a key node of shell development, and it should provide useful clues to explore essential questions concerning molluscan shell evolution, i.e., whether conchiferan and polyplacophoran shell plates are homologous , 6, 19. engrailed in the girdle field is another shared feature between polyplacophorans and conchiferans [pax2/5/8 is observed in the girdle field of the chiton Acanthochitona crinita [The common expression of hiferans , 37, 42. crinita  and the  crinita . TherefoHowever, it is still too early to say that the aforementioned data support the homology of polyplacophoran and conchiferan shell fields. Researchers revealed different cell lineages for polyplacophoran and conchiferan shell fields, arguing against such homology , 44–46. gbx is common expressed in the epithelium responsible for spicule/shell development in a aplacophoran, a polyplacophoran and a bivalve conchiferan [Given the well-supported phylogeny of mollusks comprising of Aculifera and Conchifera , 48, polchiferan .Taken together, it could be concluded that some common features of shell fields are revealed among aculiferans, between polyplacophorans and conchiferans, or between aculiferans and conchiferans, but they are far from sufficient to certify any issues regarding the homology of shell fields, that of shell plates, or that of shell plates and spicules. Compared to the extensive researches in conchiferans, studies on the development of aculiferan sclerotized structures are limited, and further investigations are required to make better comparisons to explore molluscan (shell) origin and evolution.Finally, it is notable that the development of the eighth shell plate and aesthete represents other essential aspects of polyplacophoran shell development. Given that the polyplacophoran shell field contains only the anlagen for the first seven shell plates, other manners of development may be employed for the eighth one. The sensory aesthete canal system refers to a branching canal system embedded in polyplacophoran shell plates. It is believed to be evolutionarily important due to the observation of similar structures in the shell plates and/or sclerites of ancient aculiferans and their presumptive stem lineages , 49, 50.A. rubrolineata, namely, the inner and outer girdle fields showing a concentric organization and the centrally located, alternatively arranged plate fields and ridges. Molecular patterns are revealed for each type of tissues, including common F-actin aggregations in the outer girdle field and the plate fields (with different patterns), and engrailed expression in the inner girdle field and the ridges. These results provide a detailed description regarding the structure of the polyplacophoran shell field on both molecular and cellular levels. The dynamics of different molecules revealed the ontogenesis of related tissues and provide clues for exploring the underlying regulatory mechanisms. Further studies are required to elucidate the roles of each type of cells, as well as the mechanisms modulating their specification and organization, which will help understand the formation and evolution of polyplacophoran shells and spicules.Four types of tissues were recognized from the shell field of the polyplacophoran mollusk A. rubrolineata  were collected from intertidal rocks in Qingdao, China. After transferred to the lab, each individual was placed in a 100-ml plastic cup filled with fresh seawater. In reproductive seasons (June–August), a proportion of individuals spawned within approximately 3 h after the transferring, and each type of gametes was thus collected. Sperm was added to oocyte suspension for artificial fertilization, and zygotes were cultured in filtered seawater (FSW) at 25 °C in an incubator. The developmental stages were referred to as hpf.The adults of Trochophore larvae hatched after 8.5 hpf. A few abnormal larvae were neglected in most circumstances; when their numbers could not be neglected, healthy larvae were collected from the upper half of the water column at around 10 hpf. The larvae older than 60 hpf could be induced to metamorphose by supplying plastic sheets coated with the algae collected from the rock that their parents inhabited. We found that the timepoints to start metamorphosis were very different for different individuals. This prevented us from investigating the development during and after metamorphosis in detail, but indeed allowed us to collect a few such samples. Live samples at varied developmental stages were recorded using an Olympus CKX53 inverted microscope.2, fixed in 4% paraformaldehyde (PFA)  or 2.5% glutaraldehyde, and stored in methanol or PBSTw , as described previously [At desired developmental stages, the larvae were anesthetized by adding 1 M MgCleviously .v/v = 1:1, once) and isopentyl acetate (twice). They were then submitted to critical-point drying (with liquid CO2), coated by gold, and observed using a scanning electron microscope (Hitachi S-3400N).Samples were gradually dehydrated to ethanol, and successively transferred to a mixture of ethanol and isopentyl acetate . After curing, samples embedded in resin was sectioned into 1 μm sections. Although we stained the samples with two dyes, we found eosin staining was washed out in subsequent treatments, the samples were thus only stained dark blue.Fixed samples stored in PBSTw were successively treated with PBSTx (1X PBS plus 0.5% TritonX-100) and 0.1% BSA in PBSTx for 5 min each. Then, the samples were stained in 0.1 μM TRITC (tetramethylrhodamine)-conjugated phalloidin  at 4 °C overnight. After washing with PBSTw, the samples were mounted in glycerol and observed under a confocal laser scanning microscope (ZEISS LSM 710). In the negative control group, phalloidin was not included in the staining solution, and this yield no signals, indicating no autofluorescence of the larvae that may influence with the results.In CLSM, Z-stack projections were applied in most circumstances. When doing this, the optical sections were carefully selected to not include the deep sections containing very strong staining in the larval muscle. This strategy helped reveal very important structures, such as the F-actin aggregations in plate fields  was included in the reverse primer. After electrophoresis and cleaning, the PCR product was used as the template to generate the digoxin-labeled probe through in vitro transcription.Whole mount in situ hybridization using a probe targeting the shell-formation gene eviously . In brieSamples stored in methanol were rehydrated to PBSTw. For acetylation, rehydrated samples were transferred to TEA buffer (1% triethanolamine in PBSTw), and then treated with 0.3% acetic anhydride in TEA buffer for 5 min. After that, acetic anhydride was added to a final concentration of 0.6% and the samples were incubated for additional 5 min. After wash with PBSTw, samples were digested with 50 μg/ml protease K in PBSTw for 20 min at room temperature. Post-fixation was performed by incubating samples with 4% PFA for 2 h at room temperature. After pre-hybridization in hybridization buffer  at 65 °C for 2–5 h, specimens were transferred to hybridization buffer containing 1 ng/μl denatured probe, and incubated at 65 °C overnight. Post-hybridization washing was also performed at 65 °C, including successively washing with washing solution , 2 × SSCT  and 0.2 × SSCT . Then, the specimens were rinsed in PBSTw at room temperature and incubated with the blocking solution (PBSTw containing 0.5% blocking reagent (Roche)) at room temperature for 2 h. Antibody staining was performed by incubating the samples with blocking solution containing 1/5000 alkaline phosphate-conjugated Fab fragments of a sheep anti-digoxigenin antibody (Roche) at 4 °C overnight. After extensively washing with PBSTw, the specimens were incubated with NBT/BCIP (Roche) for color development. After that, samples were post fixed with PBSTw containing 3% PFA and 1% glutaraldehyde to avoid the fading of staining in subsequent manipulations. Stained samples were mounted in glycerol and observed under a Nikon 80i microscope.A. rubrolineata larvae were frequently used in the in situ hybridization experiments in our lab and signals were never observed in control groups, we only investigated control groups using samples at 20, 36 and 48 hpf in the present study, and no staining was observed.A sense probe was used as the negative control. Due to the limit of samples at some developmental stages, and since All experiments were repeated at least twice and they generated the same results. In each assay, we examined multiple individuals from the same batch of samples: at least 10 in SEM and phalloidin staining (under CLSM) and at least 20 in in situ hybridization. All revealed highly consistent results. Very few samples (no more than 5%) showed somewhat inconsistent results due to obviously abnormal or slowed development .Additional file 1: Figure S1. 3D projections using different subset of optical sections. The two figures are derived from the different subsets of the same CLSM file. As shown in the top right inserts, panel a is derived from the subset of optical sections that does not include muscle staining, while all sections were used in panel b. It is clearly that the inclusion of muscular tissues in the 3D projection (b) strongly affects the recognition of F-actin stripes in the shell field (compare to a). Figure S2. Organization of different parts of the shell field revealed by CLSM. Serial optical sections revealed the details of outer girdle field (a–d) and central region . In a–d, superficial nuclei were only observed for the cells lacking F-actin aggregations, which are alternatively arranged with the cells possessing the F-actin tubes. In e–j, despite the deep locations of the nuclei in plate fields (arrows) compared to those of ridges (arrowheads), the apical sides of plate field cells were expose to the surface. Figure S3. Dorsal view of a 24-hpf larva. In this larva, evident depression could be observed accross the central region of posterior ridges along the midline, as highlighted by the arrow.


Cryoconite holes are small ponds present on the surface of most glaciers filled with meltwater and sediment at the bottom. Although they are characterized by extreme conditions, they host bacterial communities with high taxonomic and functional biodiversity. Despite that evidence for a potential niche for anaerobic microorganisms and anaerobic processes has recently emerged, the composition of the microbial communities of the cryoconite reported so far has not shown the relevant presence of anaerobic taxa. We hypothesize that this is due to the lower growth yield of anaerobes compared to aerobic microorganisms. In this work, we aim at evaluating whether the anaerobic bacterial community represents a relevant fraction of the biodiversity of the cryoconite and at describing its structure and functions. We collected sediment samples from cryoconite holes on the Forni Glacier  and sequenced both 16S rRNA amplicon genes and 16S rRNA amplicon transcripts at different times of the day along a clear summer day. Results showed that a relevant fraction of taxa has been detected only by 16S rRNA transcripts and was undetectable in 16S rRNA gene amplicons. Furthermore, in the transcript approach, anaerobic taxa were overrepresented compared with DNA sequencing. The metatranscriptomics approach was used also to investigate the expression of the main metabolic functions. Results showed the occurrence of syntrophic and commensalism relationships among fermentative bacteria, hydrogenothrophs, and consumers of fermentation end products, which have never been reported so far in cryoconite.IMPORTANCE Recent evidence disclosed the presence of a potential niche for anaerobic microorganisms and anaerobic processes in supraglacial sediments (cryoconite), but a detailed description of the structure and functions of the anaerobic population is still lacking. This work used rRNA and mRNA sequencing and demonstrated that anaerobes are very active in these environments and represent a relevant albeit neglected part of the ecosystem functions in these environments. Cryoconite holes are small water-filled ponds with sediment at the bottom that are present on the surface of most glaciers during the ablation season. Although they are characterized by extreme conditions such as low nutrient content, permanently low temperatures, and high solar irradiance, they host microbial communities with high taxonomic and functional biodiversity . It has In this paper, we aim at testing the hypothesis that the anaerobic bacterial populations represent a relevant fraction of the biodiversity of the cryoconite and are metabolically active. We hypothesized that the analysis of 16S ribosomal RNA transcripts (rRNA) might better detect active but slow-growing bacterial populations compared to the most common analysis of 16S ribosomal DNA amplicon genes (rDNA), thus allowing a more accurate description of the anaerobic microbial populations and their ecosystem functions. For our study, we collected cryoconite samples from four cryoconite holes on the Forni Glacier  and sequenced both 16S rDNA and 16S rRNA. In addition, to investigate a possible diurnal turnover in the active bacterial community, we collected samples from each hole at different times of the day along a clear summer day. These cryoconite holes were stable during the sampling day without changes of shape or removal of sediments by meltwater. Finally, on a subset of samples, we also used shotgun metatranscriptomics sequencing to investigate the expression of the main metabolic functions along the day. Cryoconite holes are optimal model ecosystems for these studies because of their simplicity such as stable temperatures (0.1 to 0.5°C) and a short and truncated trophic web and are an example of shallow reservoirs maintaining permanent water and allowing sunlight penetration to the bottom .The Venn diagrams in F1,14 ≥ 12.70, false discovery rate P [PFDR] ≤ 0.009; Fig. S1 in the supplemental material), revealing higher values of the Shannon index and lower values of the Gini index in rRNA than in rDNA samples. However, the same pattern was not observed for the number of ASVs . No variation was observed in the alpha-diversity indexes according to sampling time  and cryoconite hole  in any analysis.Results of this analysis showed that both at the genus and the ASVs levels, the inclusion of rRNA in the analyses led to an increase of the detected taxa compared with rDNA only. The highest percentage of genera was present in rRNA samples (52%), the smallest fraction in rDNA samples (14%), and an intermediate fraction was shared (34%) between rRNA and rDNA samples. At the ASV level, we obtained similar results. The rRNA samples contained the highest percentage of ASVs (58%), while the lowest was shared between rDNA and rRNA (9%), while an intermediate percentage was found in rDNA only (33%). Generalized linear models (GLMs) showed that both Shannon and Gini indexes varied significantly with the genetic material (rDNA or rRNA)  except for holes I and III, which did not differ . Variation partitioning analysis showed that the type of genetic material explained more variance than the hole identity  performed on amplicon data, including the type of genetic material, the cryoconite hole, and time as predictors, confirmed that the structure of the observed communities differed between rDNA and rRNA . In addiidentity .2-metabolizing, methylotroph, lithotrophic, anoxygenic phototrophic, oxygenic phototrophic, and fermentative bacteria according to the genetic material (rDNA or rRNA). Paired t tests on samples with both rRNA and rDNA data showed that aerobic genera were more abundant in rDNA than in rRNA , while obligate anaerobic genera were more abundant in rRNA than in rDNA . All the other metabolisms investigated did not differ significantly between rDNA and rRNA (PFDR ≥ 0.052). The taxonomic classification of the metabolic groups is reported in Fig. S2. The results of quantitative PCR (qPCR) quantification of 16S rRNA and narG (Table S1) indicated that the copy numbers of the 16S RNA gene were in the orders of magnitude of 109 to 1010 per gram of cryoconite and their transcripts varied between 1.3 × 106 and 8.9 × 106 copy number per gram due to the lower extraction yield of RNA compared with DNA. narG gene copies resulted to be 1 order of magnitude lower than their respective 16S rRNA genes; conversely, narG transcript copies exceeded by 3 orders of magnitude the copy numbers of the 16S RNA transcripts.In the amplicon data sets, the main energy metabolic traits were investigated, including all the classified genera with a relative abundance >0.1%. Shotgun metatranscriptomics was carried out on the 4 samples collected from hole II. Glacial ecosystems gained special attention in the last few years due to their unique physical characteristics, unique biodiversity, and rapid global disappearing . The bio10–In this study, we used both DNA- and RNA-based 16S rRNA gene sequencing to investigate the biodiversity of cryoconite holes and to accurately describe the composition of the anaerobic bacterial communities.Proteobacteria , Actinobacteria, Chloroflexi, Acidobacteria, Cyanobacteria, and Bacteroidetes , and their DNA was probably masked by that of the more abundant but inactive taxa. This result is consistent with those already reported by Anesio et al.  showed that the oxic part of the cryoconite on Forni Glacier measured in the field is restricted to the upper layer (ca. 400 μm), which represents a minor fraction of the cryoconite mass, consistently with the results published by Poniecka et al. . Clostridium members are obligate anaerobic bacteria that have been already reported in Forni cryoconite at high abundance at the beginning of the melting season  and, likely, also with methanogenic archaea, which are not included in the amplicon data. However, the transcripts of some genes of the methanogenesis pathway have been annotated in the shotgun metatranscriptome, namely, formylmethanofuran dehydrogenase subunit (fmdA), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (ftr), methenyltetrahydromethanopterin cyclohydrolase (mch), and heterodisulfide reductase (hdrB2) , which suggest that methanogenic archaea were present in our samples, but were not detected because we did not use primers specific for archaea.g season . Propionediments . MembersParacoccus), acetogenesis , and sulfate reduction (Desulfovibrio). The presence of methanogenic archaea can be inferred also by the detection in the rRNA data of methylotrophs such as Methylobacterium, Methylotenera, and Methylocaldum , which can feed on the methane produced by methanogenic archaea. Candidatus Cloacamonas members are uncultured bacteria mainly found in anaerobic digesters but also described in anoxic soils and sediments .Interestingly, the hydrogenotrophic bacteria classified in rRNA data displayed different energy metabolisms: denitrification .The shotgun metatranscriptomics allowed describing the metabolic function associated with the active bacterial community of the cryoconite during the daytime. The results showed that different carbon and energy metabolisms are expressed throughout the day . AerobicnarG, nirK, and norB) were transcribed as already reported in the cryoconite of a Chinese glacier (dsrA) transcripts were detected , in disagreement with the amplicon rRNA data where sulfate reducers have been detected. This inconsistency could be explained by the higher coverage of amplicon sequencing compared with shotgun sequencing.mRNA sequencing confirmed that anaerobic respiration actively occurred in cryoconite holes, in agreement with rRNA amplicon data. However, the detected functional gene transcripts were restricted to denitrification. Indeed, the complete denitrification pathway genes  was observed at all sampling times. Indeed, Cyanobacteria and algae were the most active CO2 fixators. However, according to the taxonomic affiliation of rblcL, Proteobacteria were the most active primary producers at night and at 1:30 pm, thus suggesting that chemolithoautotrophy could be active. Despite ammonia oxidizers (Nitrosomonas and Nitrosospira) have been detected among the chemolithotrophic genera in the rRNA amplicons, the expression of amoA (ammonia monooxygenase A subunit) was negligible at any time. Conversely, the transcription of narG, which codes for nitrate reductase alpha subunit, was found. This protein is involved in the oxidation of nitrite to nitrate, thus its presence is in agreement with the detection of Nitrospira in the rRNA amplicons. Carbon monoxide oxidation to CO2 was previously hypothesized as a possible lithotrophic metabolism in cryoconite due to the high abundance of carbon-monoxide dehydrogenase (coxML) (aprA and dsrA), only soxA was transcribed . The presence and activity of sulfur-oxidizing bacteria were supported by the presence of Sulfuricella among the classified genera in both rDNA and rRNA amplicons. However, sox genes are also harbored by anoxygenic phototrophs  resulted affiliated with alpha and betaproteobacteria  and are known to be photoheterotrophic, thus using the organic matter as a carbon source  . Howevertotrophs .2-consuming genera have been retrieved both under oxic and anoxic conditions. Consistently, shotgun metatranscriptomics data showed that hydrogenase (hyaB) genes were actively transcribed (2 fixation was due to nonphototrophs (4.30 a.m. and 1.30 pm). This might indicate that hydrogen oxidation might provide alternative reducing power to photosynthesis. This report of shotgun metatranscriptomics in cryoconite, therefore, shows that different microbial taxa contribute to the same ecosystem functions  along the day and primary productivity was supported by both photo- and chemosynthesis. This functional redundancy likely enhances ecosystem stability in such extreme environments.As previously mentioned, hydrogen seems to be actively utilized by chemolithotrophs. Indeed, different Hnscribed . This trIn summary, our study showed that only the use of integrative molecular approaches may facilitate the study of microbial biodiversity and functions in the cryosphere. For example, rare microbial taxa play an important role in cryoconite environments ; however3C6H5O7, and 70 g of (NH4)2SO4, DEPC-H2O for a final volume of 100 mL  at four different times  on 24 July 2018. The cryoconite holes were located on the ablation tongue of the Forni Glacier  at 2,670 m above sea level. To preserve the RNA content, cryoconite was immediately mixed with an RNA preservative solution (1:5 volume ratio) prepared with 4 mL of EDTA 0.5 M, 2.5 mL of 1 M Naf 100 mL  and storTotal DNA was extracted from 0.5 g of each cryoconite sample with the FastDNA Spin for Soil kit  according to the manufacturer’s instructions. A detailed description is presented in reference https://lpsn.dsmz.de/): obligate anaerobic, H2-metabolizing, methylotroph, lithotrophic, anoxygenic phototrophic, oxygenic phototrophic, and fermentative bacteria.The extracted RNA was retrotranscribed with the RevertAid First Strand cDNA Synthesis kit  using random primers. V5 to V6 region of the 16S rRNA gene was amplified and sequenced using both extracted DNA and cDNA as the templates, as previously reported . Sequenchttps://github.com/najoshi/sickle).The total DNA-free RNA from each sample was treated with the MICROBExpress Bacterial mRNA Enrichment kit (Ambion) and 8 μL of the depleted RNA were retrotranscribed with the RevertAid First Strand cDNA Synthesis kit  using random primers. Shotgun sequencing was applied to cDNA by HiSeq Illumina  using a 150-bp × 2 paired-end protocol on one lane. The paired-end reads were quality-trimmed  using Sickle (mer from 40 to 99 (with a step of 5). Predicted genes were inferred from contigs with Prodigal  included 331 forward (5′-TCCTACGGGAGGCAGCAGT-3′) and 797 reverse (5′-GGACTACCAGGGTATCTAATCCTGTT-3′). narG encodes the subunit alpha of respiratory nitrate reductase and was used as a marker gene for nitrate reduction, while forward primer narG 1960F (5′-TAYGTSGGGCAGGARAAACTG-3′) and reverse primer narG 2050R (5′-CGTAGAAGAAGCTGGTGCTGTT-3′) were used to amplify a 109-bp fragment of the aforementioned gene.Quantification of the number of copies of 16S rRNA and molecular markers for denitrification (narG) was performed through qPCR on PCRmax ECO 48 real-time PCR system  on both DNA and cDNA. The analyses were carried out on three samples. The primer set used to target the rRNA 16S 466-bp fragment  with 0.3 μM forward and reverse primer final concentration. Each assay included a control without the template. To confirm the specific amplification of the target gene, melting curves were obtained by heating amplicons.The amplification cycle for the 16S rRNA gene consisted of an initial DNA denaturation at 95°C for 4 min, followed by 40 cycles of denaturation at 95°C for 15 sec, primers annealing at 60°C for 30 sec, and extension at 72°C for 15 sec. Fluorescence was recorded at the end of each elongation step. A final cycle  was performed to obtain dissociation curves.The same steps conducted at different temperatures were used to quantify the genes targeted for microbial anaerobic metabolism. The amplification cycle consisted of initial DNA denaturation (95°C for 4 min), followed by 40 cycles of denaturation (95°C for 15 sec), primers annealing (53°C for 30 sec) and extension (72°C for 15 sec). Fluorescence was recorded at the end of each elongation step. Dissociation curves were produced as follows: 95°C for 15 sec, 55°C per 15 sec, and 95°C for 15 sec.To quantify the PCR products, standard curves based on threshold values were obtained through the amplification of 10-fold dilutions series of standard DNA obtained through the generation of a plasmid containing the target regions using each primer set.post hoc tests (Tukey method), correcting P values for multiple statistical tests according to the false discovery rate (FDR) procedure (Singletons (ASVs present in one sample only) were removed from the ASVs table. Statistical analyses were performed on a nonrarefied data set with R 3.5.1  with therocedure .https://www.ncbi.nlm.nih.gov/sra/PRJNA816448).Sequence data were submitted to NCBI SRA database, BioProject number 816448 (


BMAL1) gene is an important circadian clock gene and previous studies have found that certain polymorphisms are associated with type 2 diabetes in adults. However, it remains unknown if such polymorphisms can affect fasting glucose in children and if other factors modify the associations.The brain and muscle Arnt-like protein-1 . Adjusting for the same confounders, rs3816358 was shown to be significantly associated with fasting glucose . Furthermore, a significant interaction between rs3789327 and nutritional status on fasting glucose was identified (Pinteraction = 0.009); rs3789327 was associated with fasting glucose in the overweight/obese subgroup , but not in non-overweight/non-obese children.After adjusting for age, sex, body mass index (BMI), physical activity, and unhealthy diet, GG genotype carriers of BMAL1 polymorphisms were significantly associated with the fasting glucose level in children. Additionally, the observed interaction between nutritional status and BMAL1 supports promoting an optimal BMI in children genetically predisposed to higher glucose level.BMAL1 were associated with fasting blood glucose levels in children. Additionally, there was a significant interaction between nutritional status and BMAL1 affecting fasting glucose levels.Polymorphisms in the essential circadian clock gene BMAL1 rs3789327 was associated with fasting glucose only in overweight/obese children.This finding could bring novel insights into mechanisms by which nutritional status influences fasting glucose in children. A Chinese and Danish cross-population twin study demonstrated a high genetic influence on fasting glucose levels, with heritability ranging from 55 to 71%.6 However, blood glucose levels are influenced by both environmental and genetic factors, and the gene–environment interaction is also very important.8It is estimated that the prevalence of diabetes in the global adult population in 2021 is approximately 10.5% (536.6 million), which is projected to rise to 12.2% (783.2 million) by 2045.9 Brain and muscle Arnt-like protein-1 (BMAL1) is an important component of the circadian clock in mammals and controls the oscillations of the circadian rhythm. It has been shown that disruptions of circadian rhythm oscillations in glucose metabolism are involved in the pathogenesis of type 2 diabetes.10 Sadacca et al. demonstrated that BMAL1 is essential for normal insulin secretion and glucose homeostasis in pancreatic beta cells.11 Additionally, an experimental study suggested that obesity and diabetes may reduce the rhythmic expression of clock genes in the liver and adipose tissue.12 Population studies about BMAL1 gene polymorphisms and risk of type 2 diabetes were controversial and all conducted in adults.15 Until now, the relation between BMAL1 gene polymorphisms and fasting glucose levels in children is not clear. Therefore, the purpose of this study was to investigate the relationship between BMAL1 gene polymorphisms and fasting glucose levels in Chinese children.Many aspects of physiology and behavior, such as the daily rhythms of food intake, metabolism, and, more specifically, glucose metabolism are regulated by the circadian clock system.BMAL1 polymorphisms could interact with nutritional status affecting fasting glucose has not been demonstrated before. Thus, we also investigated the interaction between BMAL1 gene polymorphisms and nutritional status on fasting glucose levels in Chinese children.In addition, whether Using a cluster sampling method, a total of 1019 children aged 10–15 years from 3 middle schools in Changsha city, Hunan Province, China were included. The study was conducted in 2019. Seven participants were excluded for invalid questionnaire data. Sixty-five participants were excluded for absence of glucose phenotype data or genotype data. Therefore, a total of 947 participants were included in the present study. The study was approved by the Medical Ethic Committee of Hunan Normal University. Written informed consents was obtained from all participants and their parents.17 Dietary behaviors included fried chips/cakes/cookies and soft drinks. Participants were given the option of “eating/drinking in the past 7 days” (Yes), or “not eating/drinking in the past 7 days” (No). According to the “Dietary Guidelines for Chinese School-Age Children (2022)”, the physical activity was divided into “<1 h/day” and “≥1 h/day”.18Height and weight were measured according to standard protocols. Peripheral venous blood sample were collected under an overnight fasting condition. Then the fasting plasma blood glucose was measured using GOD-PAP method with the auto analyzer OLYMPUS AU400 with a standard protocol. For the fasting requirement, parents of the included children were informed one day before the blood collection by text message, and also during the blood collection, the nurses confirmed the fasting status of the participants. Covariates, including demographic characteristics and lifestyle factors, such as physical activity and dietary behaviors, were investigated by questionnaire.19 Also, the stratified association between BMAL1 gene polymorphisms and blood glucose level were analyzed with multiple linear regression models in children with different glucose status .According to fasting blood glucose, the definition of prediabetes was 5.6–6.9 mmol/L and diabetes was ≥7.0 mmol/L in Chinese children.2). Nutritional status of children was defined according to Working Group of Obesity in China (WGOC) criteria, of which the age and sex-specific cut-off points are the 85th and 95th percentiles of BMI .20Body mass Index (BMI) was calculated by weight divided by square of height .Selection of the BMAL1 gene polymorphisms was based on previous literature regarding population studies on the BMAL1 gene and cardiometabolic risk factors. Polymorphisms with significant association with cardiometabolic risk factors were selected. Then, we used the 1000 genome database to identify the MAF of the SNPs in East Asians and only SNPs with MAF > 0.05 were included in the present study. Finally, four SNPs from the 23 Matrix-assisted laser desorption/ionization time of flight mass spectrometry  was used for the genotyping of BMALI polymorphisms.16 Genotyping was performed by investigators who were blind to the participants’ phenotypes. All the call rates of the genotyping were above 98%  was calculated using the formula FST = (P1 − P2)2/[(P1 + P2) × (2 − (P1 + P2))].24P1 is the effect allele frequency of a gene polymorphism in the 1000 Genomes Project database of European ancestry and P2 indicates the gene frequency of the effect allele in this study. FST reflects the ancestral differences in the same gene polymorphism between the two populations. FST between 0 and 0.05 indicates a small ancestral difference; A range of 0.05–0.15 indicates a moderate ancestral difference; 0.15–0.25 indicates a large ancestral differences; a FST > 0.25 indicates very large ancestral difference.25 Descriptive statistical analysis was used to analyze the general demographic characteristics, genotypes and allele frequencies of the participants. T tests were used for continuous variables and Chi-square test was used for categorical variables. The best genetic models were selected according to the Akaike Information Criterion (AIC).26 Multivariable linear regression analysis was conducted to analyze the association between gene polymorphism and fasting glucose, and the regression coefficients (b) and standard error (SE) were presented. Model 1 is the crude model; for Model 2, we added sex, age, and BMI as covariates; and Model 3 included sex, age, BMI, physical activity, and unhealthy diet (including soft drink and consumption of fried chips/cakes/cookies) as covariates. Considering 4 SNPs of BMAL1 were selected in the present study, we adjusted multiple testing for Bonferroni correction (P < 0.05/4 = 0.0125). SPSS for Windows  was used for statistical analysis.Hardy–Weinberg equilibrium was checked with the Chi-square test. P < 0.05). The prevalence of overweight and obesity in boys (24.6%) was significantly higher than that in girls (15.9%) (P = 0.001). For age, BMI, glucose status, unhealthy diet , and genotype frequency of BMAL1 polymorphisms, no significant sex differences were found (P > 0.05). The ancestral differences in BMAL1 gene polymorphisms between European population in the 1000 Genomes Project database and our population are all small . In the additive genetic model, we found that rs3816358 polymorphism A allele was significantly associated with fasting glucose level after adjustment for sex, age, BMI, physical activity, and unhealthy diet . No significant associations between rs10832020 or rs7950226 and fasting glucose levels were found (P > 0.05). However, none of the polymorphisms were significantly associated with glucose level after correction for multiple comparison (P < 0.05/4 = 0.0125). We also analyzed the association between BMAL1 gene polymorphisms and blood glucose level in children with different glucose status , and neither in normal blood glucose group nor in children with prediabetes/diabetes participants significant association between the four SNPs of BMAL1 and blood glucose level was found . In the overweight/obese subgroup, rs3789327 polymorphism GG genotype carriers had significantly higher fasting glucose than the AG/AA genotype carriers , but no significant association was examined in the subgroup who were not-overweight/non-obese. No significant interactions were observed for the rs10832020, rs7950226, and rs3816358 polymorphisms of BMAL1. The adjusted fasting glucose levels stratified by different genotypes and nutritional status are shown in Fig. A significant interaction between rs3789327 and nutritional status on fasting glucose was found  in adults.22 In addition, Evans et al. found that the T allele of rs3816358 is significantly related with later sleep onset time  objectively measured actigraphy in the Elderly in the United States.27 Previous studies reported that sleep onset time is significantly related with glucose-related phenotypes, such as diabetes, obesity, and fasting blood glucose, since later bedtime is likely to be associated with shorter sleep duration, leading to disruption of glycemic control or glycemic rhythmicity.29 But in the present study, the sleep traits of children were not investigated, which limited us to explore the potential mediation effect of sleep onset time in the association between rs3816358 and fasting blood glucose. Future studies could be conducted to further characterize this mediation effect.The finding of an association between the 32 This is the first study detecting association between rs3789327 polymorphisms and glycemia related phenotypes. However, the significant association was not significant after correcting for multiple testing. The findings should be interpreted with caution, and larger sample size studies and functional studies are needed to verify these results.In addition, our study shows that the GG genotype of rs3789327 might be a risk genotype for high fasting glucose levels in children. Previous studies have focused on different readouts, demonstrating associations with myocardial infarction, multiple sclerosis, and depression.BMAL1 gene polymorphisms and blood glucose levels in normal blood glucose and prediabetes/diabetes participants, no significant results were found. Several previous studies have shown that BMAL1 gene polymorphisms are associated with the risk of developing type 2 diabetes in adults.15 However, studies have noted that BMAL1 gene polymorphisms are not associated with the occurrence of type 2 diabetes in Japanese33 and African-American34 cohorts. In contrast to the findings of our study, Pappa et al. showed that rs7950226 polymorphism of BMAL1 gene is associated with susceptibility to gestational diabetes in Greek pregnant women.14 However, this might be due to the different age, ethnic diversity and varying environmental conditions of the study population. Furthermore, different genetic risk factors might exist in different population with different ethnicity.The current study did not detect significant associations between rs7950226/rs10832020 and fasting glucose. In addition, we also analyzed the association between BMAL1 acts an essential component of the circadian oscillation, which drives the daily rhythms of physiology and behavior.35 In pancreatic β cells, BMAL1 plays a key role in mediating insulin secretion, exocytosis and metabolism.36 According to previous functional studies about clock genes and glycemic phenotypes, the possible link between BMAL1 and fasting glucose levels might be mitochondrial dysfunction.39Mitochondrial dysfunction is increasingly considered to be the main driver of pancreatic β cell failure in the pathogenesis of diabetes mellitus, and the loss of normal β cell function is the core factor of impaired insulin secretion in diabetes mellitus.40Regarding potential mechanism underlying the associations between BMAL1 gene polymorphisms and fasting glucose, it is noteworthy that BMAL1 gene was reported before. However, recent studies have found an association between BMAL1 and indicators of obesity and obesity is closely associated with elevated fasting glucose in children.42 Being overweight or obese might amplify the genetic susceptibility of unfavorable glucose level for specific genotype carriers of BMAL1 gene polymorphism. Previous studies have reported that glucose-related phenotypes (Type 2 diabetes) which are attributed to genetic predisposition can be significantly different in people with different nutritional status.44 The identified interaction of rs3789327 with nutritional status on glucose in our study is of clinical interest, considering that mounting evidence have shown that fasting glucose levels in childhood are significant predictors for diabetes and other related cardiometabolic risk factors in adulthood.46 Mechanistically, studies have demonstrated that BMAL1 can activate CRY gene expression in conjunction with the CLOCK gene when BMAL1 levels are high.47 The degradation of CRY1 induces gluconeogenesis and maintains blood glucose levels, but high fat intake accelerates the degradation of autophagy CRY1 and contributes to the development of obesity-related hyperglycemia. Furthermore, a recent study observed that BMAL1 overexpression can enhance circadian clock function and β cell function, thus enhancing GSIS and systemic glucose metabolism in the context of diet-induced obesity.48 In short, the above-mentioned findings could imply that BMAL1 gene polymorphisms and nutritional status have an essential role in affecting fasting blood glucose levels in children.Furthermore, results of the current study demonstrated a significant interacting effect between nutritional status and rs3789327 on fasting blood glucose levels. In overweight/obese individuals, GG genotype carriers had significantly higher levels of fasting blood glucose than AA/AG genotype carriers, which was not detected in children without overweight/obesity. To the best of our knowledge, no gene–nutritional status interaction of BMAL1 gene polymorphisms and results have been controversial in different ethnic populations. Genetic studies during childhood, a period when environmental risk factors  are relatively minimal, could increase the possibility to detect a genetic risk factor that might otherwise be masked, therefore contribute to elucidate the etiology of abnormal glucose and early onset diabetes. Fasting blood glucose levels in children are closely related to genetic factors and an early onset of diabetes also dictates the development and severity of chronic complications. Children are also the ideal population for genetic association study of blood glucose.One strength of our study is that it focused on children. Previous studies have generally set their focus on the association of cardiovascular metabolic risk factors in adults with BMAL1 gene but there are far more known SNPs in this gene. Secondly, studies in adults have observed controversial results regarding the association between BMAL1 gene and risk of diabetes in different ethnic groups. Therefore, further studies on children of other ethnic groups are needed. Finally, we did not investigate information about puberty among the study participants, so we were unable to explore the potential effect of puberty on fasting blood glucose levels.However, there are also some limitations in our study. We only selected 4 representative SNPs of the BMAL1 rs3789327 and rs3816358 polymorphisms are significantly associated with fasting blood glucose levels in Chinese children. We also found that rs3789327 was associated with fasting blood glucose in overweight/obese children, but not in non-overweight/non-obese children. In addition, nutritional status and rs3789327 had an interacting effect on fasting blood glucose levels. Our finding highlights the importance of promoting healthy nutrition, especially in children with a genetic susceptibility to higher fasting glucose. Thus, these findings might also contribute to the development of early prevention strategies for elevated blood glucose in children.In conclusion, our study demonstrates that Supplementary Tables


Multiple myeloma (MM) is an incurable hematologic malignancy originating from clonal plasma cell proliferation within the bone marrow, predominantly affecting older individuals. While anemia serves as a diagnostic criterion for MM, it often ameliorates upon achieving disease remission. Iron metabolism parameters have emerged as potential prognostic indicators in MM. Notably, physical exercise has been established to influence iron metabolism. This study aimed to assess alterations in serum iron, ferritin, and transferrin concentrations, as well as leukocyte gene expression, in MM patients undergoing a six-week cycle of Nordic walking training. Thirty patients divided into an exercise group  and a control group  completed the study protocol. Blood samples were collected at baseline, after three and six weeks of training, and after nine weeks. Serum ferritin, transferrin, and iron concentrations were measured, along with the leukocyte expression of genes. Additionally, serum oxidative damage marker levels were determined. Following the Nordic walking training cycle, a declining trend in serum ferritin concentrations was observed. Intracellular mRNA levels of genes associated with iron metabolism were positively influenced by the training regimen, indicating the potential impact of this physical activity on gene expression and ferritin concentrations. Although positive trends were noted, extended training periods might be requisite for significant changes. To conclude, moderate-intensity exercise induces favorable shifts in the analyzed parameters among MM patients, potentially influencing disease progression. Consequently, Nordic walking training is a safe recommendation for MM patients, though sustained training beyond six weeks could be necessary for notable effects on iron metabolism factors. Multiple myeloma (MM) is a hematologic malignancy originating from plasma cells found in the bone marrow. This disease accounts for approximately 10 to 15% of all diagnosed hematologic malignancies and approximately 1–2% of all cancer cases [PCBP1 (poly(rC) binding protein 1), PCBP2 (poly(rC) binding protein 2), FTH1 (ferritin heavy chain 1), FTL (ferritin light chain), and TFRC (transferrin receptor 1), which are involved in iron homeostasis, are induced by stress stimuli, among which are physical exercise. Oxidative stress and inflammation parameters are usually increased after exercise. This effect is particularly noticeable after performing intense physical exertion, such as running a marathon [PCBP1, PCBP2, FTL, and FTH lead to understanding changes in regulation of iron economy in white cells and could be independent of plasma indicators in healthy and ill people [Iron homeostasis is of great importance in patients with MM, as they often experience anemia (approximately 75% of patients), which is one of the diagnostic criteria for this disease ,3. It ismarathon  and Finamarathon . Howeverl people .Oxidative stress is one of the main factors which influences iron metabolism and the expression of genes associated with it. The level of oxidative stress and oxidative damage increases with age  and in tFree radical-induced damage also plays a significant role in MM. Patients with MM have been shown to exhibit high levels of oxidative stress compared to healthy individuals. This is associated with decreased activity of antioxidant enzymes in these patients and increased levels of lipid peroxidation products, protein oxidation, and DNA damage . TherefoWalking with poles, also known as Nordic walking, is a safe form of physical activity recommended for both older individuals and patients with hematologic malignancies. In patients with MM in remission, it has been shown to have a beneficial effect on disease parameters  as well The aim of this study was to assess the impact of a six-week cycle of Nordic walking training on iron metabolism parameters and the expression of related genes in patients with MM. Additionally, this study aimed to evaluate the effects of oxidative stress by measuring the serum concentrations of products of oxidative modifications of macromolecules.−2 and the average CG was 28.77 ± 4.18 kg∙m−2. No statistical differences between the selected groups were indicated. All patients enrolled in the study were in remission stage of MM and had undergone the autologous hematopoietic stem cells transplantation (ASCT) procedure at least six months before the beginning of the study and achieved remission. Among NW patients, IgG myeloma was diagnosed in eight subjects , IgA—in six patients , and in one subject light chains myeloma was detected. In CG, four patients each were diagnosed with IgG kappa and IgG lambda myeloma. IgA kappa MM type was diagnosed in five participants and IgA lambda in two.The age of the study group performing Nordic walking trainings cycles  was 63.1 ± 8.4 years, and the time since diagnosis of MM was 39.8 ± 13.8 months. In the control group , these parameters were 63.5 ± 3.6 years and 36.3 ± 16.6 months, respectively. The average BMI of the NW group was 29.12 ± 4.01 kg∙mThe white blood cells count, red blood cells count, and hemoglobin concentrations in all time points are shown in Significant differences between groups in iron, transferrin, and transferrin saturation (TfS) (Cohen’s d—medium to large effect) were observed, resulting from the lower concentrations obtained in the control group. Ferritin concentration was higher in the control group and no correlation between iron and ferritin was found in both groups. The first 3 weeks of training caused a slight decrease in iron, transferrin, and TfS, but not ferritin concentration. During the next 3 weeks of training sessions, further decreases in iron concentration and TfS were recorded, while transferrin remained unchanged. A slight increase in ferritin serum concentration was observed at the same time. However, 6 weeks of NW training caused statistically significant changes only for iron concentration. In the control group, iron and TfS slightly increased, but ferritin slightly decreased, especially after 6 weeks from the start of the experiment.Two-way ANOVA showed significant differences in time, row factor, and interaction between groups. It was observed that 3 weeks after the end of the experiment, iron, ferritin, and transferrin concentrations were slightly lower than at baseline only in the NW group. Cohen’s d showed that 6 weeks of applied training caused lower differences between groups for all parameters compared to baseline; however, this effect did not last up to 3 weeks after intervention.The results obtained in both groups in all four time points for macromolecule damage indicators are presented in BaselineNo differences in 3NT A, 4HNE B, and 8dEffects of NW trainingIt was observed that 3-NT A and 4HNChanges in 8-dHG C and 8-iDifferences between groupsA significant difference was observed after 3 weeks of training in 3-NT concentration A; howeveThe 4HNE B concentDifferences between groups in 8dOHG C relied Significant differences between baseline and after 3 weeks of the end of the experiment concerned 8-isoPGFa2 D which wThe reference values for these oxidative stress parameters are not clearly determined, but based on literature data, the following ranges found in young healthy individuals can be established: 8-OHdG—16.95 ± 10.66 ng/mL , 8-isoPGFTH, FTL, TFRC, PCBP1, and PCBP2 genes expression in all four times in both group points are shown in Changes in intracellular BaselineFTH and FTL mRNA were slightly higher in the NW group  that disease-free survival was significantly shorter in individuals with serum ferritin concentrations above 2100 μg/L, and overall survival was three times longer in patients with ferritin concentrations at diagnosis below 900 μg/L [FTL and FTH genes in leukemic cells also increased in patients undergoing chemotherapy, similar to other acute-phase proteins, including ferritin. In our study, comparable mRNA levels of the investigated genes were found, similar to those of healthy individuals in the study by Borkowska et al. [Increased expression of 900 μg/L . In the a et al. . In summFTL and FTH mRNA levels showed a tendency towards decreased expression of these genes after 3 and further after 6 weeks of NW training , but this effect did not persist 3 weeks after the end of the experiment, which further encourages conducting studies with a longer duration of NW activity. This decrease was accompanied by an increase in WBC, which further confirms the observed trend.Nordic walking is an activity with well-documented beneficial effects on various biochemical parameters of blood, including iron metabolism ,28,29. IPCBP1 and PCBP2 are iron chaperones that deliver iron to ferritin, the iron storage protein [FTH and FTL mRNA expression. Indirectly, this may indicate an increase in the iron pool [The observed changes in serum Fe and gene expressions may be associated with a decrease in the labile iron pool (LIP), and therefore they should be considered beneficial, as free Fe ions are susceptible to oxidation and participate in the Fenton reaction, generating free radicals. An increase in LIP leads to an increase in ferritin levels in cells. According to the literature,  protein . In our ron pool .High iron stores are associated with an increased risk of developing various non-communicable diseases, including cancer. Iron overload is also linked to a significant increase in the risk of infection in patients undergoing auto-HSCT procedures, which are often used in the treatment of MM . In a stTFRC mRNA were different from changes in serum. A clear trend of decreased expression of this gene was observed after 3 weeks of training, which persisted until the sample collection after 6 weeks, followed by an increase to initial values during the follow-up period, indicating a lasting effect .In our own study, no changes in serum transferrin concentrations, which is responsible for delivering iron to erythroblasts , were obIn patients with MM, there is an increase in oxidative stress levels, which is particularly evident during the treatment of the disease . Ansari Changes in 3-NT and 4-HNE concentrations showed that Nordic walking training induced a decrease in the concentration of these indicators . Moreover, these changes were observed also 3 weeks after the training period (follow up). Changes in 8-OHdG and 8-isoPGFa2 were ambiguous, and no effect of exercise was observed for 8-OHdG, but there was a tendency to increase in 8-isoPGFa2. Similar results were obtained by Kortas et al.  in respoThe study group consisted of 30 patients with MM recruited among the patients of the Hematology Clinic at the Jagiellonian University Medical College in Krakow. The patients were randomly assigned to one of two groups: the Nordic walking training group  and the control group, which has not been subjected to training intervention . For random assignment to groups, a permuted block randomization procedure (ratio 1:1) was used. Each participant drew an opaque envelope containing an assignment for one of the groups. CG patients were asked not to change their physical activity habits during the project period. Patient flow diagram is shown in Each patient received a daily supplementation of 2000 U of vitamin D3 and 1000–1500 mg of calcium carbonate. Additionally, in accordance with the prevailing guidelines of the International Myeloma Working Group , intraveInclusion in the study was based on a detailed medical interview and meeting the inclusion criteria . The parPatients in the NW group underwent a moderate-intensity Nordic walking training cycle (60–70% of HRmax) for a period of 6 weeks. Venous blood samples were collected from all participants at baseline (I—before the start of the project), in the middle of the training cycle before the training session (II—3 weeks), after the completion of the project (III—6 weeks), and 3 weeks after the completion . Venous blood samples were collected from the participants by a qualified laboratory diagnostician using a vacuum system (BD Vacutainer). Each time, three tubes were collected: one with an anticoagulant (EDTA) and two containing a clot activator.FTH1 (ferritin heavy chain 1), FTL (ferritin light chain), TFRC (transferrin receptor), PCBP1 (poly(rC)-binding protein 1), PCBP2 (poly(rC)-binding protein 2), primers with the following 5′-3′ sequences were used :In detail, the methods and primers applied in this study were previously described by Grzybkowska et al. . LeukocyFTH1TCCTACGTTTACCTGTCCATGForward primer: CTGCAGCTTCATCAGTTTCTCReverse primer: FTLGTCAATTTGTACCTGCAGGCCForward primer: CTCGGCCAATTCGCGGAAReverse primer: TFRCTGCAGCAGTGAGTCTCTTCAForward primer: AGGCCCATCTCCTTAACGAGReverse primer: PCBP1AGAGTCATGACCATTCCGTACForward primer: TCCTTGAATCGAGTAGGCATCReverse primer: PCBP2TCCAGCTCTCCGGTCATCTTTForward primer: ACTGAATCCGGTGTTGCCATGReverse primer: TUBB—tubulin beta class 1 (used as the reference gene)TCCACGGCCTTGCTCTTGTTTForward primer: GACATCAAGGCGCATGTGAACReverse primer: FTH1 (NM_002032), FTL (NM_000146), TFRC (NM_001128148), PCBP1 (NM_006196), and PCBP2 (NM_001128913) was calculated using qRT-PCR.The sequence of primers has been designed by the authors with the use of the Primer3 web specificity and has been screened in silico via the UCSC genome browser. The relative mRNA expression of The routine parameters of iron metabolism in serum were analyzed in a medical diagnostic laboratory. The concentrations of ferritin and transferrin were determined using an immunoturbidimetric method on the Cobas 8000 analyzer . The concentration of iron in the tested samples was measured using a colorimetric method on the Cobas 8000 analyzer .The blood collected for biochemical analysis (in a tube containing coagulation activator) was centrifuged for 10 min at 4 °C and 2500 rpm. The resulting serum was pipetted into Eppendorf-type tubes and stored in a low-temperature freezer at −80 °C until further analysis. The assessment of oxidative damage parameters  was performed using an immunoenzymatically method (ELISA test).The training sessions were conducted outdoors by a qualified Nordic walking trainer during the spring and summer seasons. The intensity of the training was set at 60–70% of the individual’s maximum heart rate (HRmax), calculated using the Nes formula . The intThe health training program, lasting 6 weeks, consisted of 18 training sessions held 3 times a week in the morning, with each session lasting approximately 60 min. Each training session was divided into three parts: 5-min warm-up (low intensity), main part lasting 45 min (moderate intensity), 10 min of cool down exercises. During the main part, exercises with poles were performed, focusing on maintaining proper technique. The walking distance was progressively increased with each training session.p values paired or unpaired . The normality of the distribution was assessed using the Shapiro-Wilk test. For variables with a normal distribution, a two-way ANOVA analysis was performed, while for non-normally distributed variables, the Friedman test was used. The effect size was calculated with the use of Cohen’s D coefficient. All statistical analyses were conducted using GraphPad Prism 9 software .For genes expression, the delta Tc method was applied . CalculaA decreasing trend in serum ferritin concentrations after Nordic walking training has been observed in patients with MM in remission taking part in this study, suggesting that the short duration of the training may not be sufficient to induce significant changes in ferritin concentrations.Intracellular changes in mRNA levels of genes related to iron metabolism showed a tendency towards decreased expression after Nordic walking training cycles, indicating an influence of the training on these genes.In conclusion, the results presented in this study suggest that the applied type of training may have beneficial effects in patients with MM. The moderate intensity exercise intervention applied in our study did not cause an increase in oxidative damage of macromolecules, which is a favorable effect. However, longer periods of training may be necessary to achieve significant favorable changes as observed in cited studies in healthy subjects. The present study provides further evidence that Nordic walking training is a safe form of physical activity for patients with MM in the disease remission phase, and as such should be recommended to patients. In our opinion, any treatment which induces decreases in genes and proteins associated with iron metabolism could be treated as positive and extend the period of remission, wherein a similar tendency refers to oxidative stress parameters. Thus, Nordic walking training is suitable for patients with MM; however, it is worth considering a longer period than 6 weeks of training.


Introduction: Currently, there are limited accessible and cost-effective biomarkers for preclinical Alzheimer’s disease (AD) patients. However, the apolipoprotein E (ApoE) polymorphic alleles can predict if someone is at high (e4), neutral (e3), or low (e2) genetic risk for developing AD. This study analyzed electroencephalogram (EEG) reports from individuals with various ApoE genotypes, aiming to identify EEG changes and patterns that could potentially serve as predictive markers for preclinical AD progression.Methods: Participants aged 64-78 were selected from the patient database at an outpatient neurology clinic. Genotype studies were performed to determine ApoE status, followed by EEG analysis to identify any apparent trends. A case-control design was used, categorizing participants into cases  and controls (e3e3). EEG recordings were compared between the groups to identify potential differences in EEG characteristics, including abnormal temporal slowing, frequency, and ApoE genotype association.Results: Among 43 participants, 49% demonstrated evidence of abnormal temporal slowing on EEG. Of these, 48% displayed focal left temporal slowing, and 52% displayed bilateral temporal slowing. The right-sided temporal slowing was not observed. Among participants with abnormal slowing, 95% exhibited theta frequency (4-8 Hz) slowing, while only 4.8% displayed delta frequency (0-4 Hz) slowing. Among participants with the ApoE4 allele, 61.5% demonstrated evidence of abnormal slowing, compared to 43.3% without it. Furthermore, the presence of an ApoE4 allele was associated with a significantly higher proportion of males (54%) compared to those without it (13%) (p=0.009).Conclusions: Although we did not find a statistically significant difference in temporal EEG slowing among different ApoE genotypes, our findings suggest a potential association between temporal slowing on EEG and the presence of an ApoE4 allele in individuals with preclinical AD. These observations highlight the need for further exploration into the potential influence of the ApoE4 allele on EEG findings and the utility of EEG as a complementary diagnostic tool for AD. Longitudinal studies with large sample sizes are needed to establish the precise relationship between EEG patterns, ApoE genotypes, and AD progression. Alzheimer’s disease (AD) is a progressive neurodegenerative disease and the leading cause of dementia. In 2018, around 50 million individuals were affected, with projections indicating that this number could surpass 150 million by 2050 ,4. DiagnThe pathophysiology of AD is complex and multifactorial. One theory suggests that it involves the accumulation of proteins beta-amyloid and hyperphosphorylated tau in the brain, leading to neuronal cell death . StudiesDespite extensive research, there is no curative treatment for AD. However, early detection and diagnosis allow for the initiation of disease-modifying therapies to slow the progression of the disease. Therefore, early detection, timely diagnosis, and appropriate management of AD are imperative ,12. PrevThis study was sanctioned by the University of Hawaii Institutional Review Board (protocol number: 2019-00054), and written informed consent was obtained from all subjects as appropriate.Study design A case-control design was used to investigate specific EEG findings in preclinical AD patients with known ApoE status. Participants were categorized into two groups: cases and controls. The control group consisted of individuals with an e3e3 ApoE genotype, while the other ApoE genotype groups  constituted the cases. A comprehensive analysis was conducted to evaluate the impact of different ApoE genotypes on participant characteristics. This analysis aimed to identify potential differences in gender distribution, age, and the presence of temporal slowing on EEG among participants with different ApoE genotypes. Statistical analysis was made using Fisher's exact test and the Kruskal-Wallis rank sum test. The significance level was set at p<0.05. All calculations were performed in R .To investigate the relationship between the presence or absence of ApoE4 and its association with temporal slowing on EEG, a separate analysis was conducted. Statistical analysis was made using Fisher's exact test to compare the presence of temporal slowing on EEG between participants with and without ApoE4. To examine the differences in gender distribution and age between participants with and without ApoE4, Fisher's exact test and Welch's two-sample t-test were used. A separate subgroup analysis was performed for individuals who demonstrated bilateral slowing. Fisher’s exact test was used to evaluate differences in ApoE4 presence between individuals with independent and synchronous bilateral slowing. The significance level was set at p<0.05. All calculations were performed in R (version 4.3.1).Subjects Participants (n=43) were recruited from a larger cohort of patients (n=127) screened for ApoE status at an outpatient neurology clinic from 2019 to 2022. Individuals were identified as having preclinical AD using specific inclusion and exclusion criteria. Inclusion criteria required participants to be 18 years of age or older, display no clinical signs or symptoms of AD, and have at least one marker of AD pathology. Exclusion criteria encompassed a prior diagnosis of either AD or mild cognitive impairment and a history of major structural brain disease, stroke, transient ischemic attack, traumatic brain injury, or seizures. To ensure cognitive health, a minimum MMSE score of 28 was required. ApoE genotype was determined through genetic testing using standard molecular biology techniques.EEG recordingsEEG recordings were conducted at an outpatient neurology clinic using the standard 10-20 measuring system and gold disc electrodes affixed with Ten20 conductive paste . The Cadwell Arc recording hardware and software were utilized, along with 22 standard scalp leads and a two-lead, single-channel electrocardiogram. Scalp electrode impedances were reduced to 10 kΩ or lower. The primary montage used was the "longitudinal anatomic bipolar" with the option for rederivation to a different montage. Patients were reclined comfortably during the recordings, which lasted between 25 and 30 minutes.Reactivity was measured with eye opening and closing while patients were awake and alert. Photic stimulation was performed at rates of 1, 3, 6, 12, 15, 18, 20, and 30 Hz, for 10 seconds each, with 10 seconds in between. Hyperventilation was performed for three minutes unless medically contraindicated. The remaining study time was dedicated to quiet recording to facilitate drowsiness or sleep. EEG reports were analyzed using visual analysis and manual counting by a fellowship-trained neurophysiologist.In this study, "slowing" was defined as a reduction in the frequency of the brain wave pattern waveforms compared to the expected normal range for the participants' age and cognitive function. In this study, we specifically looked at slowing in the temporal region. Slowing was categorized into two frequency ranges: delta (0-4 Hz) and theta (4-8 Hz). Slowing was also categorized as intermittent or continuous. Slowing was further characterized as focal or bilateral. Bilateral slowing was categorized as independent or synchronous. Additionally, we recorded whether participants were able to attain sleep during the EEG study, the presence of findings in wakefulness, drowsiness, or both and the presence of epileptiform changes.Patient characteristicsThis study involved 43 participants Table , consistEEG featuresAmong the 43 participants, 21 (49%) displayed evidence of abnormal temporal slowing on EEG  has been linked to reduced EEG functional connectivity in various brain regions of AD patients compared to heterozygous ApoE4 carriers and controls . MoreoveThe ApoE4 genotype is thought to contribute to neurodegenerative processes, negatively impacting cortical activity in both healthy individuals and those with AD . Since tThe statistically significant difference in the proportion of males between individuals with and without an ApoE4 allele in their genotype was an interesting finding. While unintentional, this discrepancy could have potentially influenced the study outcomes, as AD can present differently in males and females . One stuThere are several limitations of this study. Notably, the relatively small sample size for certain genotypes may have affected the statistical power of the analysis. Therefore, drawing definitive conclusions from these specific subgroups should be done with caution. For instance, the e2e4 genotype was represented by only one participant, and the e4e4 genotype was represented by only four participants. A larger sample size would have allowed for more robust statistical analyses and increased generalizability. However, given that this pilot study serves as a preliminary exploration, a smaller sample size was appropriate. Another limitation was that the EEG analyses were made visually and, therefore, subject to the reader’s interpretation. A standardized approach using quantitative EEG analysis could provide more objective and reliable results. While the practicality of EEG usage is advantageous, relying solely on visual analysis can introduce potential biases. Future studies should consider utilizing both visual EEG analysis and quantitative EEG analysis to enhance the validity and reliability of their findings. Finally, accurately identifying individuals at the preclinical stage of AD versus those at more advanced stages of AD was challenging due to the subjective nature of the diagnosis, which can vary among different providers. It is possible that some individuals included in the study were at more advanced stages of the disease, potentially influencing the study results.Biomarkers for timely detection and diagnosis of AD before the onset of permanent memory loss are crucial. Although no statistically significant relationship was observed between temporal EEG slowing and the ApoE4 allele in individuals with normal cognitive function, the trends suggest that the ApoE4 allele may influence EEG findings. Future investigations are needed to accurately assess the predictive value of EEG as a readily available, noninvasive, and inexpensive tool for screening and identifying individuals at high risk of developing AD. These studies should examine longitudinal data in a larger patient population with various ApoE genotypes and examine the appearance of EEG temporal slowing at different stages as patients progress from preclinical, to mild, to severe AD. Altogether, these findings could enhance our understanding of the pathophysiology of disease progression and the potential window of opportunity for intervention with disease-modifying therapies.