When delivered to live cells, ZF-tMeCP2engages the NCoR/SMRT corepressor complex, selectively represses transcriptionfrom methylated promoters, and colocalizes with heterochromatin inmouse primary cortical neurons. We also report that efficient nucleardelivery of ZF-tMeCP2 relies on an endosomal escapeportal provided by HOPS-dependent endosomal fusion. The Tat conjugateof MeCP2 (Tat-tMeCP2), evaluated for comparison,is degraded within the nucleus, is not selective for methylated promoters,and trafficks in a HOPS-independent manner. These results supportthe feasibility of a HOPS-dependent portal for delivering functionalmacromolecules to the cell interior using the cell-penetrant mini-proteinZF5.3. Such a strategy could broaden the impact of multiple familiesof biologic-based therapeutics.The vast majority of biologic-based therapeutics operatewithinserum, on the cell surface, or within endocytic vesicles, in largepart because proteins and nucleic acids fail to efficiently crosscell or endosomal membranes. The impact of biologic-based therapeuticswould expand exponentially if proteins and nucleic acids could reliablyevade endosomal degradation, escape endosomal vesicles, and remainfunctional. Using the cell-permeant mini-protein ZF5.3, here we reportthe efficient nuclear delivery of functional Methyl-CpG-binding-protein2 (MeCP2), a transcriptional regulator whose mutation causes Rettsyndrome (RTT). We report that ZF- The cell-permeantmini-protein ZF5.3 efficiently deliversthe therapeutically relevant transcriptional regulator MeCP2 to thenucleus of model cells and primary neurons while preserving proteinfunction. Yet progress toward this goal has been exceptionallyslow. Most delivery strategies8 are inefficient,9 rely on degradation-prone molecular scaffolds,10 operate via undefined or multifarious mechanisms,11 and evaluate activity using amplified or indirectassays.12The efficient delivery of proteins andnucleic acids to the cytosoland internal organelles remains a significant and unmet challengein an era exploding with novel therapeutic strategies. The vast majorityof FDA-approved biologics are delivered via injection, and operatewithin serum, on cell surfaces, or within endosomal vesicles. Thosethat do operate in the cytosol16 is taken up by the endosomalpathway and released efficiently intothe cytosol and nucleus of live cells,14 alone and when fused to certain protein cargos. Proteins successfullydelivered using ZF5.3 include the self-labeling protein SNAP-tag,9 the metabolic enzyme argininosuccinate synthetase,16 the proximity labeling tool APEX2,9 and a nanobody-based degrader.17 These proteins differ in molecular weight, stoichiometry,isoelectric point, and the presence of bound cofactors. In all casesevaluated,16 the protein that reached the cytosol wasfully intact as judged by Western blot analysis of isolated cytosolicfractions, and the delivery efficiencies were 2–10-fold9 higher than seen with canonical or cyclic peptides.18 Mechanistic studies confirm that ZF5.3 relieson the endocytic pathway to reach the cell interior,13 and that endosomal escape into the cytosol demands a functionalhomotypic fusion and protein sorting (HOPS) complex.15We showed previously that the mini-proteinZF5.319 Mutations in the X-chromosome-linked MECP2 gene cause Rett syndrome (RTT), a severe and incurable neurologicaldisorder that disproportionately affects young girls.20 Many potential RTT treatments are under development,21 but no disease modifying treatment yet exists.Two features of RTT etiology render therapeutic development especiallychallenging. The first is that more than 850 different mutations inthe MECP2 gene22 accountfor >95% of classical RTT cases;23 thisfeature complicates approaches based on gene-editing.26 The second is toxicity caused by overexpression of MeCP2; this featurecomplicates approaches that rely on gene delivery.31 Since 2007, several studies have demonstrated thatrestoring 70–80% of the native levels of MeCP2 protein couldalleviate neurological symptoms of RTT, restore neuronal signaling,and greatly improve the survival rate in male and female MeCP2-deficientmice.34 These rescue experiments support the hypothesis thatdose-dependent, nuclear delivery of functional MeCP2 protein couldprovide a novel treatment modality. Although the concentration ofMeCP2 varies between cell types, its primary function is to regulategene transcription. MeCP2 can repress gene expression by engagingthe NCoR/SMRT corepressor complex in a methylated DNA-dependent manner.35 By interacting with CREB1, it can also functionas a transcription activator.36 In orderto be effective, MeCP2 protein must reach the nucleus intact, transcriptionallyactive, and in the high nanomolar to low micromolar concentrationrange.37Methyl-CpG-binding-protein 2 (MeCP2) is an abundantnuclear proteinexpressed in all cell types, especially neurons.10) was reportedto deliver a MeCP2-GFP fusion protein to the nucleus with low efficiency(2–10%) after cells were treated with high concentrations (20μM) of the fusion.38 A conjugatebetween MeCP2 and residues 47–57 of the HIV-1 transactivatorof transcription protein Tat (Tat-MeCP2) reached the interior of humanfibroblasts40 and primary mouse neurons10 as determinedby confocal microscopy and Western blots, reversed histone hyperacetylation,and interacted with the partner protein HDAC1.41 In this case, however, long incubation times (>12 h) were needed,the established nuclear concentration was low (∼4 nM),39 and significant degradation was observed.10Previous efforts to deliver MeCP2protein were largely ineffective,in large part because of low delivery efficiencies and significantlevels of cargo degradation. A cell-permeable nanobody functionalizedwith a cyclic arginine-rich peptide  (referred to henceforth as tMeCP2)is shorter  but mirrors MeCP2 in its interactionswith methylated DNA and the NCoR/SMRT complex, and Mecp2-null male mice display a near-normal phenotype upon expression ofMeCP2(ΔNC).28 We generated fusionproteins containing a single copy of ZF5.313 or Tat47–5742 followedby the complete sequence of tMeCP2 mutation that reduces specific DNA binding44 and is seen commonly in RTT patients.23 The second is P302L tMeCP2, which has a diminishedability to engage the TBLR1 subunit of the NCoR/SMRT repressor complex.45 All tMeCP2 variants were expressedin E. coli, purified to >95% homogeneity,and characterized using Western blots and LC/MS  in the absence of DNA  contains 484 amino acids (52 kDa) 1A.28 MeCf tMeCP2 1A. Each nd LC/MS . VariantLC/MS  interacts with methylatedDNA in the heterochromatin region43 whilethe C-terminal transcriptional-repressor domain (TRD) domain engagesvarious partners such as the NCoR1/2 corepressor complex and CREB1.47 To establish whether the tMeCP2 proteins studied here retain these functions in vitro, we measured their affinities for methylated andnonmethylated DNA oligonucleotides using a fluorescence polarizationassay and used immunoprecipitation methods to assess interactionswith corepressor proteins in lysates (tMeCP2 interacts with methylated DNA with a KD of 15 nM and a 15-fold preference for methylated versusnonmethylated DNA. ZF-tMeCP2 interacts with methylatedDNA with a KD of 55 nM and a 6-fold preferencefor methylated DNA. The conjugate of Tat47–57 and tMeCP2 (Tat-tMeCP2) interacts with bothmethylated DNA (KD = 5 nM) and nonmethylatedDNA (KD = 25 nM) more favorably than tMeCP2 and ZF-tMeCP2 with a 5-fold preferencefor methylated DNA. As expected, ZF-tMeCP2(T158M)binds poorly to both methylated (KD =210 nM) and nonmethylated (KD = 1.1 μM)DNA when compared to tMeCP2. Although the KD describing the interaction of tMeCP2 with methylated DNA has not previously been determined, reportedvalues for full-length MeCP2 fall in the range of 36–130 nMwith a 2–33 fold preference for methylated DNA.49An important function of MeCP2 is to modulategene transcription from methylated promoters. lysates 1B,C. Flu lysates 1B. tMeCPtMeCP2 proteins in vitro, we used an affinity pull-downassay to evaluate their interactions with the NCoR/SMRT corepressorcomplex in nuclear lysates of NIH3T3 cells.28 Lysates47 were incubated overnight at4 °C with 1.5 μM of ZF-tMeCP2, Tat-tMeCP2, or tMeCP2; ZF-tMeCP2(P302L) was used as a negative control. Each tMeCP2 variant was extracted from the lysates using streptavidin-coatedbeads, and the identities and relative levels of bound NCoR/SMRT subunits were evaluated using Western blots. Taken together, these two in vitro assays confirm that purified ZF-tMeCP2 retainsthe core functions of MeCP2: selective recognition of methylated DNAand the ability to engage the NCoR/SMRT corepressor complex.To further probe the function of purified rn blots1C. ThesetMeCP2 variant and specifically how much proteintrafficked to the nucleus, the site of MeCP2 function. Human osteosarcoma(Saos-2) cells were incubated for 1 h with rhodamine-tagged tMeCP2-Rho, ZF-tMeCP2-Rho, Tat-tMeCP2-Rho, or ZF-tMeCP2(T158M)-Rho atconcentrations between 0.5 μM and 2 μM -Rho also showed evidence of intranuclearfluorescence at concentrations as low as 1 μM, while cells treated tMeCP2-Rho did not, even at 2 μM concentration. Whenvisualized as 3D z-stacks, cells treated with ZF-tMeCP2-Rho, Tat-tMeCP2-Rho, and ZF-tMeCP2(T158M)-Rho differed in intranuclear localization -Rhoshowed aggregated rhodamine signal in small discrete regions resemblingnucleoli. This observation aligns with previous reports that truncationof the entire MBD or T158 M mutation resulted in MeCP2 relocalizationto the nucleolus.53Next, we made use of three fluorescence-basedmethods and two model cell lines to evaluate the overall uptake ofeach and 2 μM 2A. When ed cells. Saos-2 lization . Cells ttMeCP2-Rho conjugate in Saos-2 and CHO-K1 cells  or ZF-tMeCP2(T158M)-Rho (1.6-fold) -Rho,and Tat-tMeCP2-Rho reach the nucleus more efficiently(2.0–2.3-fold) than tMeCP2-Rho, with localizationefficiency increasing in the order tMeCP2-Rho <Tat-tMeCP2-Rho < ZF-tMeCP2-Rho∼ ZF-tMeCP2(T158M)Rho (We next used fluorescence correlation spectroscopy(FCS) to quantitativelytrack the cytosolic and nuclear distribution of each K1 cells . FCS is  cytosol , as esta cytosol . LocalizeCP2-Rho . At low .6-fold) 2D. At 1 158M)Rho 2D.tMeCP2 resulted in a smaller fold-improvementin nuclear or cytosolicdelivery than previously reported examples (improvements between 316 and 329 fold), the mean nuclear concentrationof ZF-tMeCP2 established in Saos-2 cells after a1 h incubation with 1 μM protein (709 ± 69 nM) is the highestintracellular concentration yet measured for a protein delivered byZF5.3.16 The high concentration of ZF-tMeCP2 that reaches the nucleus may result from the higher intrinsicpermeability of tMeCP2 itself in comparison to otherproteins when evaluated under comparable conditions .16 While further studies are needed to establish the factors that leadto high intrinsic permeability, we note that tMeCP2is characterized by both a high pI (10.78) and high levels (60%) ofintrinsic disorder as judged by CD (We note that while the conjugationof ZF5.3 to ed by CD .vide infra), we also evaluated the trafficking of each tMeCP2-Rhovariant to the cytosol and nucleus of CHO-K1 cells using confocalmicroscopy -Rhowas too high to be reliably measured by FCS.55 Although the total fluorescence of intact cells measured using flowcytometry does not reliably quantify how much material escapes fromthe endosomal pathway,9 procedures to isolateand sort nuclei on the basis of light scattering and fluorescenceare well-known.57 We wondered whether thefluorescence of nuclei isolated from cells treated with tMeCP2-Rho variants would correlate with the nuclear concentrationmeasured in intact cells using FCS. If such a correlation was observed,then flow cytometry would provide an extremely high-throughput andrapid alternative to FCS for quantifying delivery of fluorescentlytagged material to the nucleus.The nuclear fluorescenceof Saos-2 cells treated with 2 μM ZF-tMeCP2-Rho variants and 2 μM tMeCP2-Rho, andthe nuclei isolated and evaluated using flow cytometry (R2 = 0.75) with the intranuclear concentrationspreviously measured by FCS for all four Rho-tagged tMeCP2 proteins, regardless of overall delivery efficiency -Rho showed consistently low nuclearlocalization efficiencies across all treatment concentrations, asexpected since the MBD is essential for MeCP2 binding in the nucleus.58 Unfortunately, we could not repeat this experimentin CHO-K1 cells due to extremely low BacMam reagent transduction efficiency.We also note that although nuclear pore complex inhibitors have beenreported to help to avoid false positive signals from proteins leakinginto the nucleus during lysis,58 minimalrhodamine interference was observed in this experiment  and cytosolic(tubulin) proteins confirmed that nuclear fractions were free of detectablecytosol and endosome contaminations used to deliver FDA-approved mRNA vaccines–remain incompletelycharacterized. Most improvements in delivery are empirical and modest,and the involvement of endogenous protein factors has not been rigorouslyexcluded.Progress in direct protein deliveryhas been slowed by an insufficient understanding of how non-nativecargo traffics across endosomal membranes and into the cytosol. Althoughendosomal escape mechanisms used by certain viruses, such as influenzaA,61 Previous mechanistic studies indicate thatefficient cytosolic and nuclear trafficking of ZF5.3 relies on theHOPS complex, but not the analogous CORVET complex.15 We thus sought to investigate if this dependence also heldfor the ZF5.3 conjugate of tMeCP2.Two multisubunit tethering complexes play importantroles in the endosome maturation pathway. A class C core vacuole/endosometethering (CORVET) complex facilitates the fusion of Rab5 positiveearly endosomes, while the fusion of Rab7 positive late endosomesto lysosomes requires the homotypic fusion and protein sorting (HOPS)-tetheringcomplex.tMeCP2-Rho, ZF-tMeCP2-Rho, or Tat-tMeCP2-Rho for 1 h andanalyzed by flow cytometry (tMeCP2-Rho thatreached the cytosol (3.1-fold) or nucleus (4.7-fold)  decrease in the concentration ofZF-tMeCP2-Rho that reached the nucleus (2.4-fold).Even the trafficking of tMeCP2 itself was affectedby the knockdown of both VPS39 and TGFBRAP1  or CORVET (TGFBRAP1) in Saos2 cells, using a nontargetingsiRNA (RISC-free) and lipofectamine only treatment as controls 3A. The cytometry 3B and FCytometry 3C–E. Totytometry 3B. Knock.7-fold) 3D; theseTGFBRAP1 3C. It ist-tMeCP2 3E. This iciently 2E, via a15 The observation that the high nuclear deliveryefficiency of ZF5.3-tMeCP2 depends on both HOPS andCORVET, favors the former explanation and deserves further study,especially as HOPS and CORVET share 4 of 6 subunits in common.61 Identification of those molecular features that promote productiveinteraction with HOPS and/or CORVET could improve the efficiency ofother delivery strategies, including lipid nanoparticles, whose efficiencyremains <10%.64We previously proposed that HOPS may engage directlywith ZF5.3to promote escape during vesicle fusion; it may also promote traffickinginto intraluminal vesicles as a prerequisite to endosomal escape.tMeCP2 proteins delivered to the nucleus of CHO-K1cells, which express low levels of endogenous MeCP2  upon immunoprecipitation,as observed in vitro in lysates  andendosomal  contaminants  was encoded at the C-terminus ofmNeonGreen to promote its turnover and improve assay sensitivity.67 Functional tMeCP2 variantsthat reach the nucleus should selectively repress transcription fromcells transfected with the methylated SNRPN promoter, leading to lessmNeonGreen fluorescence relative to controls. By contrast, deliveryof a trace, nonfunctional, or nonspecific tMeCP2variant will either not repress transcription or do so without selectivityfor the methylated promoter.In the nucleus, MeCP2 acts as a bridge todeliver the NCoR/SMRT complex to methylated promoters; this recruitmentrepresses transcription.er genes 4C. BrieftMeCP2, ZF-tMeCP2, Tat-tMeCP2, or ZF-tMeCP2(T158M) and the green fluorescenceemission at 530 ± 30 nm was monitored using flow cytometry. Cellstreated with tMeCP2 itself displayed high mNeonGreenfluorescence levels regardless of promoter methylation state  all showed dose-dependent decreasesin mNeonGreen fluorescence. The highest levels of methylation-specifictranscriptional repression was observed in cells treated with 5 μMZF-tMeCP2, Tat-tMeCP2, as well asZF-tMeCP2(T158M), although measurable effects wereseen at concentrations as low as 2 μM .55 Fitting the autocorrelationcurves obtained from intranuclear measurements in CHO-K1 cells witha two-component 3D diffusion equation68 identified a population of tMeCP2-Rho variantsthat diffuses freely in the nucleoplasm  and a second population that diffuses slowly , presumably because itis bound to DNA (tMeCP2 diffusesslowly (Fslow = 26.3%) is higher thanthat of ZF-tMeCP2(T158M) (16.3%) in accord with relativemethylated DNA affinities determined in vitro  is counterbalanced by the low DNA bindingpopulation; the result is no significant transcriptional repression all reached the nucleus at significantly higherlevels than tMeCP2 .69 The highest levels of methylation-dependenttranscriptional repression were observed in cells treated with ZF-tMeCP2 and ZF-tMeCP2(T158M). As a previousstudy suggested, MeCP2(T158M) is capable of binding to methylatedDNA in a protein level-dependent manner.70 At low treatment concentrations (1–2 μM), most ZF-tMeCP2(T158M) was sequestered in the nucleolus, so the effectivenuclear concentration of ZF-tMeCP2(T158M) is nothigh enough to rescue its reduced methylated DNA binding affinity.As more protein reaches the nucleus at 5 μM treatment, a higherlevel of the methylated promoter is bound and repression is observed.Taken together, these data indicate that the fusion of ZF5.3 to tMeCP2 does not interfere with the protein’s selectivitytoward methylated DNA. ZF-tMeCP2 reaches the nucleusat a concentration high enough to observe methylation-dependent transcriptionrepression. Notably, although fluorescent detection implies relativelycomparable nuclear delivery of ZF-tMeCP2 and Tat-tMeCP2, the latter is largely degraded . This delivered concentration is 2 orders of magnitudehigher than a previously reported Tat-MeCP2 conjugate that rescuedcertain RTT-related symptoms.41 Rigorous analyses verify the absence of significant nuclear degradationand the presence of MeCP2-specific activity. Multiple independentapproaches, including intact nuclear flow cytometry, a novel and generalapplication of flow cytometry, quantitatively assess delivery in anonamplified and high-throughput manner. Finally, the remarkable levelof nuclear delivery by ZF-tMeCP2 enables its potentialapplication to reverse Rett syndrome phenotypes.In this work, we address the challengesof intracellular proteindelivery by using the stable mini-protein ZF5.3 to hijack the endosomalmaturation machinery and guide the methyl-CpG-binding-protein 2 (MeCP2)into the nucleus at a therapeutically relevant concentration. A modestdose to Saos-2 cells  results in a nuclear concentrationof 2.1 × 10


To investigate the independent and collective effects of maternal folic acid supplementation or dietary folate intake on the risk of low birth weight (LBW), and to further comprehensively examine the joint associations of folic acid supplementation and dietary folate intake with LBW by various clinical subtypes.OR) and 95% confidence interval (95% CI) for single and joint associations of folic acid supplementation and dietary folate intake with LBW.Participants were recruited from Gansu Provincial Maternity and Child Care Hospital. A standardized and structured questionnaire was distributed to collect demographic factors, reproductive and medical history, occupational and residential history, physical activity, and diet. Data on pregnancy-related complications and birth outcomes were extracted from medical records. Unconditional logistic regression models were used to estimate the odds ratio , and the reduced risk was mainly seen for term-LBW , and multiparous-LBW . There were no significant associations between dietary folate intake and LBW, and there was no interaction between folic acid supplement and dietary folate intake on LBW.Compared with non-users, folic acid supplementation was associated with a reduced risk of LBW (Our study results indicated that folic acid supplementation was associated with a reduced risk of LBW, and there was no interaction between folic acid supplements and dietary folate intake on LBW. Low birth weight (LBW) can increase neonatal mortality and is associated with various infant morbidities , 2. FurtFolate is the generic term for compounds that have vitamin activity similar to that of pteroylglutamic acid and is an anti-anemic and growth factor. Folate functions as a co-enzyme in several single carbon transfers leading to the biosynthesis of purine nucleotides and deoxythymidylic acid essential for DNA and RNA synthesis .et al. found that folate deficiencies are known risk factors for LBW  were associated with birth weight , Q2 (143.24–188.55), Q3 (188.55–254.37), Q4 (≥254.37), and folic acid supplementation was classified into no more than 12 weeks and more than 12 weeks.OR) and 95% confidence interval (95% CI) for single and joint associations of folic acid supplementation and dietary folate intake with LBW and various clinical subtypes. Dose-response relationships (p for trend) were calculated based on those categorical levels. In Tables 2, 3, we adjusted for maternal age, monthly income per capita, maternal education level, smoking, maternal employment, weight gain during pregnancy, preeclampsia, cesarean section, parity, total energy intake, dietary folate intake, or folic acid supplement. In Table 4, we adjusted for maternal age, monthly income per capita, maternal education level, smoking, maternal employ, weight gain during pregnancy, preeclampsia, cesarean section, total energy intake, dietary folate intake, or folic acid supplement. In Table 5, we adjusted for maternal age, monthly income per capita, maternal education level, smoking, maternal employ, weight gain during pregnancy, preeclampsia, cesarean section, parity, and total energy intake. All analyses were performed using SAS software, version 9.4 .Pearson's chi-square tests were used to compare selected characteristics between NBW and LBW. Unconditional logistic regression models were used to estimate the odds ratio , and the risk of LBW decreased with the increasing duration of folic acid supplementation (p for trend <0.001). After stratifying by periods of folic acid supplementation, slightly significant associations were observed for those who took supplements before conception and during pregnancy  or during pregnancy only , and significant duration of dose-responses for folic acid supplements were observed for both (p for trend <0.001 and <0.001). However, this pattern is not similar to women who took supplements before conception only at all. Dietary folate intake is not associated with LBW. In addition, there was no interaction between folic acid supplements and dietary folate intake on LBW (p = 0.223).As shown in OR: 0.59, 95% CI: 0.41–0.85), but it was not presented at preterm-LBW , and the similar associations were observed for women who took supplements before conception and during pregnancy  or during pregnancy only . For dietary folate intake, there were no significant associations between term-LBW and preterm-LBW.We then analyzed the data separately for term-LBW and preterm-LBW . The proOR: 0.72, 95% CI: 0.54–0.94), but folic acid supplementation was not related to nulliparous-LBW . We further analyzed multiparous-LBW by periods of folic acid supplementation, the protective effect was significantly increased before conception and during pregnancy , but not during pregnancy only  or before conception only , and a significant duration of dose-responses for folic acid supplementation was observed before conception and during pregnancy (p for trend < 0.001), and during pregnancy only (p for trend <0.001). For dietary folate intake, there were no significant associations with nulliparous-LBW and multiparous-LBW.Depending on parity, we divided LBW into nulliparous-LBW and multiparous-LBW . A signiOur study results indicated that folic acid supplementation was associated with a reduced risk of LBW, term-LBW, and multiparous-LBW, with those risks decreasing with increasing duration of folic acid supplementation. After stratifying by periods of folic acid supplementation, similar patterns were observed for those who took supplements before conception and during pregnancy or pregnancy only. There was no interaction between folic acid supplement and dietary folate intake on LBW.The occurrence of LBW is a highly complex biologic process, and the precise protective mechanism of folic acid is still unknown. The epigenome is particularly susceptible during the early stages of embryogenesis , folate et al. found that preconception start of folic acid was associated with a decreased risk of LBW, and the start of folic acid supplementation after pregnancy recognition was also associated with a decrease of having a child with LBW  for the publication of any potentially identifiable images or data included in this article.QL and JQ designed the birth cohort study. LY, WW, and BM analyzed data and wrote the article. LY, WW, BM, BY, XH, XL, LL, XX, YCh, and YCa conducted this research. YCh and YCa had primary responsibility for the final content. All authors read and approved the final manuscript.This study was financially supported by BiosTime Maternal and Child Nutrition Health Research Projects of China CDC Maternal and Child Health Center (No. 2019FYH002), in part supported by Gansu Provincial Science and Technology Department Grant (No. 21JR1RA043), the Health Research Projects (No. GSWSKY-2019-98), and the Key Research and Development Program (No. 20YF8WA095). The funders had no role in the design, analysis, or writing of this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Salariopsis fluviatilis from Italian geographic areas (north-western regions and Sardinia Island) so far never investigated and uses five species delimitation methods in order to shed further light on the Mediterranean phylogeography of this fish and give a more comprehensive scenario of its taxonomic status. Our findings clarified the phylogeographical patterns of the northern Italian populations of Salariopsis fluviatilis sensu stricto, considering the Po River basin and some Tyrrhenian basins of the Liguria region on the other side of the Ligurian Alps. The dispersal pathways of the island of Sardinia were also investigated suggesting that patterns of genetic structuring for this species are probably linked to Pleistocene glacial and interglacial periods. Results obtained supported previous studies in evidencing the occurrence of a species complex for Salariopsis fluviatilis with at least three taxonomic units: Salariopsis fluviatilis sensu stricto, Salariopsis cf. fluviatilis diffused in the Middle East and a further taxonomic entity from the Iberian Guadiana River basin whose tributaries flow in the Atlantic Ocean. For what concerns Salariopsis fluviatilis sensu stricto, two divergent groups of populations were reported in the present study, being the first diffused in western Mediterranean areas and the second in western Adriatic and eastern Mediterranean areas.The present study provides new molecular data on populations of the freshwater blenny Salariopsis (Blenniidae) comprises freshwater blenny fish that inhabits Mediterranean Sea, Black Sea, and north-east Atlantic areas. Three species were formally described to date: Salariopsis fluviatilis. S. economidisi, and S. atlantica. In this study, 103 individuals were collected from different Italian regions  and analyzed using the mtDNA Control Region and the ribosomal 16s gene. We aimed (i) to depict the phylogeographic patterns of S. fluviatilis in northern Italy and Sardinia and (ii) to compare the genetic structure of Italian samples with those from other Mediterranean regions. Results obtained showed the presence of a well-supported genetic structuring among Italian S. fluviatilis populations, shedding new light on the phylogeographic patterns of northern Italian populations of S. fluviatilis sensu stricto across the Ligurian Alpine ridge and the Sardinia Island-mainland dispersal patterns. Furthermore, our species delimitation analysis was consistent in supporting results of previous research about the presence of genetic differentiation among S. fluviatilis, evidencing: (i) a large group of S. fluviatilis sensu stricto that includes two sub-groups , (ii) one group comprising populations from the Middle East of a taxonomic entity corresponding to Salariopsis cf. fluviatilis, and (iii) one group of Iberian individuals from the Guadiana River.The genus  Salariopsis fluviatilis  (Actinopterygii: Blenniiformes: Blenniidae), is a small benthic fish that inhabits river basins and lakes around the Mediterranean Sea. It belongs to the recently described genus Salariopsis Vecchioni, Ching, Marrone, Arculeo, Hundt & Simons, 2022, proposed by Vecchioni and co-authors [Salaria Forsskål, 1775. Nowadays, the genus Salariopsis includes freshwater species whose distribution is across the eastern Atlantic coasts (Morocco), the Mediterranean Sea, and the Black Sea [The freshwater blenny, is Asso, 801 , endemic to Lake Trichonis in Greece [Salariopsis atlantica Doadrio, Perea & Yahyaoui, 2011, endemic to the Seboui river basin in Morocco [In this context, in addition to  Morocco .S. fluviatilis shows, within the genus, the widest range of distribution, its populations are generally small and highly localized [Salariopsis species, which show confined  or even point-like  distributions.Although ocalized ,21,22,23S. fluviatilis is highly susceptible to several anthropomorphic disturbances and to global environmental change effects. Nevertheless, even though it has been considered vulnerable or endangered in several countries and reported in Appendix III of the Bern Convention, S. fluviatilis has been evaluated by the International Union for Conservation of Nature (IUCN) as data deficient (IUCN 2013), due to a lack of data on its abundance and population trend.Similar to other freshwater fish living in fragmented habitats, S. fluviatilis [Salariopsis in the Mediterranean area, with inferences reported also for S. fluviatilis. Wagner et al. [S. fluviatilis populations: (1) Middle East ; (2) Guadiana (Guadiana basin with the Zùjar and Esteras rivers that flow on the European Atlantic coast); (3) Algeria-Verde (in southern Spain and Algeria with the Verde River and Oued Boughzazene); (4) Occidental basin , (5) North Oriental basin , and (6) the Island of Crete (Greece). The results of Wagner et al. [Salariopsis fluviatilis. Remarkably, based on the results of two methods of species delimitation, Belaiba et al. [S. fluviatilis from the Middle East Mediterranean region should be regarded as a differentiated species, i.e., Salaria cf. fluviatilis. However, in light of the description of the new genus Salariopsis [Salariopsis cf. fluviatilis hereafter.Despite the potential interest of this species from phylogeographic, taxonomic and conservation perspectives, not many studies have been performed to depict the genetic structure and phylogeography of viatilis ,20,24,25r et al. , in partr et al.  also supr et al. ), suggesa et al.  suggest lations:  Middle ES. fluviatilis in the western Mediterranean [S. fluviatilis in the Mediterranean would be the product of the recent spreading of individuals from the two glacial refuges of the Iberian Peninsula and Corsica Island. However, in this study, the lack of samples from Liguria  probably affected the possibility to fully reconstruct the dynamics of colonization which involved Corsica Island. Indeed, Wagner et al. [S. fluviatilis from Corsica, highlighted the importance of focusing studies on island populations due to the peculiarity and fragility of the island’s fish fauna, to infer the biogeographical dynamics of the populations that live there. These same authors also expressed hope for extending the geographical sampling to provide a more comprehensive scenario of S. fluviatilis in the Mediterranean area.Furthermore, two main studies focusing specifically on the nuclear genetic variation of erranean ,25 suggeerranean . In partr et al. , analyziS. fluviatilis is distributed in a discontinuous way, starting from the northern regions and continuing through the Tyrrhenian side, down to Campania, Sardinia, and the Sicily islands. Isolated populations have also been found in Calabria and on the Adriatic coasts of the peninsula [S. fluviatilis and the country is characterized by different (hydro)geographic boundaries that strongly shaped its ichthyofauna with the presence of several endemic species—64 taxa are considered native to inland waters of Italy [S. fluviatilis living on Sardinia Island deserve particular attention, as this species is one of the few putative native freshwater fish known for the island [S. fluviatilis from Italian areas that are not yet under investigation. Indeed, knowledge of the genetic structure of populations from north-west Italy and the Sardinian Island could help to fill in some gaps in the mosaic of S. fluviatilis genetic distribution in the central-western Mediterranean.Focusing on Italy, eninsula ,27. Althof Italy  and amonof Italy —no wide-e island . The fole island ,32,33,34S. fluviatilis populations, sampled in Sardinia and north-west Italy  in order to shed light on the phylogeography of the populations now living in the main central-western Mediterranean islands and the north-west Italian mainland; (2) to compare the genetic structure of our samples with that of other Mediterranean regions to put them in a wider geographic framework; and (3) to make an attempt to further clarify the taxonomy of the species across its whole range of distribution by using different methods of species delimitation to evidence, if any, distinct evolutionary lineages within S. fluviatilis that deserve to be deeply investigated in the future.As a results, the goals of our paper were: (1) to depict the phylogeographic patterns of S. fluviatilis allowed us to compare the sequences obtained in the present study with many others from different geographic areas. In previous studies, the combined use of Control Region and 16s sequences has been demonstrated to be helpful in providing insights into the genetic structuring and phylogenetic history of S. fluviatilis populations [Two portions of the mitochondrial Control Region and of the 16s ribosomal gene were used as markers. The use of the mtDNA, which is maternally inherited, is considered an appropriate choice to solve intraspecific taxonomic and phylogenetic issues and to depict evolutionary lineages ,36,37. Fulations ,15,17.Salariopsis fluviatilis were caught using an electric stunner from the freshwaters of 4 different Italian regions  between February 2019 and March 2022  and immediately transferred to a recovery tank before being released. Tissues were preserved in absolute ethanol and used to perform DNA extraction. The above reported sampling method was approved by the ethics committee of the University of Sassari (Prot n. 122 770 of 7 November 2022), and its researchers led the sample collection activities during the present study.The protocol of sampling and analysis of the fish fauna of wadable lotic systems, provided by the Italian Higher Institute for Environmental Protection and Research (ISPRA) , was folSardinian samples were collected in three tributaries of the Flumendosa river , all confluent in the Alto Flumendosa man-made reservoir (originated in the middle of the last century by a dam) , the Rio Mannu di Scano Montiferro river , and the Rio Mannu di Posada river (north-east Sardinia); Ligurian samples were collected in the Entella river (east Liguria) and in the Roja river (west Liguria); samples from Piedmont were collected in the San Giovanni river, in the San Bernardino river, and in the Strona di Omegna river; the sample from Lombardy was collected in the Tartaro Fuga creek, tributary of the Oglio river.2 was set at 200 µM and 1.5 mM, respectively. PCRs were performed in a GeneAmp PCR System 9700 Thermal Cycler , programmed as follows: 1 cycle of 4 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 51 °C (for both Control Region and 16s gene), and 30 s at 72 °C. At the end, a post-treatment of 10 min at 72 °C and a final cooling at 4 °C were carried out. Both positive  and negative controls were used to test the effectiveness of the PCR protocols and the absence of possible contaminations. Electrophoresis was carried out on 2% agarose gels, prepared using 1× TAE buffer  and stained with Gel Red Nucleic Acid Stain . PCR products were purified by ExoSAP-IT  and sequenced for forward and reverse strands (by means of the same primers used for PCR), using an external sequencing core service .Total genomic DNA was isolated from a portion of fin tissue using the Macherey-Nagel Nucleo Spin Tissue Kit , following the supplier’s instructions. DNA solutions were quantified using the Nanodrop™ Lite Spectrophotometer , which showed an average yield of 29 ng/µL. A portion of the mitochondrial Control Region and rRNA gene 16s were amplified by standard PCR using the following primers: CR-F (forward) (5′-CCACTAGCTCCCAAAGCTA-3′) and CR-R (reverse) (5′-CAGGACCAAGCTTTTGTGC-3′) ; 16s Forhttps://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 16 September 2022)) and deposited in GenBank (OP675744-OP675846 for the Control Region and OP653611-OP653713 for 16s).A total of 103 newly generated sequences was aligned using the package Clustal Omega   available on GenBank to date (last update 30 September 2022) (see The Control Region and 16s 022) see .S. economidisi  and S. atlantica . Furthermore, the marine species Salaria basilisca (MW555061) and Salaria pavo (MW555062) were used as outgroups. As well, in the 16s dataset, the sequences available for the Mediterranean freshwater blenny species were also included, i.e., S. economidisi  and S. atlantica . The marine species—S. basilisca (MH724820) and S. pavo (FJ465707)—were used as outgroups , number of haplotypes (H), haplotype diversity (h), and nucleotide diversity (π) using the software package DnaSP 6.12.03  .To test the reliability of the three datasets  for taxonomic and phylogenetic analyses, the phylogenetic signal was checked by the software TREEPUZZLE  : the likThe best probabilistic model of sequence evolution was achieved using jModelTest 2.1.3  , with a www.fluxus-engineering.com) (accessed on 12 October 2022) . Transitions and transversions were equally weighted. Due to the absence of information about the possible appearance of retro-mutation events, the same weight (10) was assigned to each observed polymorphism.To infer the genetic relationships among haplotypes and to detect the possible occurrence of discrete genetic clusters, a median-joining network  was consSalaria from GenBank: they grouped 280 sequences for the Control Region (see http://tree.bio.ed.ac.uk/software/figtree/) (accessed on 18 September 2022) .S. fluviatilis sequence was checked using five different methods of species delimitation. The GMYC  model tests for a significant shift in the branching rate along an ultrametric species tree. The analysis was performed on the ultrametric species tree obtained from the Bayesian dating analyses by means of the SPLITS (SPecies LImits by Threshold Statistics) package [http://r-forge.rproject.org/projects/splits/)(accessed on 18 September 2022). Species entities were identified by means of the single threshold option, which uses a single threshold in order to specify the transition from between- to within-species branching .http://species.h-its.org/ptp/) (accessed on 18 September 2022) on the phylogenetic species trees using default options and 500,000 MCMC generations. To test the reliability of results, each run was examined for convergence by visualizing the likelihood plot: if convergence occurred, the chain should stay at high likelihood locations during the run  ) uses tha et al. ).http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html) (accessed on 18 September 2022) using the default settings. The correct species estimate was selected, according to Puillandre et al. [p = 0.001   method ie et al. , using ta et al. ).https://cran.rproject.org/) (accessed on 18 September 2022). The script partitions taxa into entities applying the fixed threshold of 2% obtained by Hebert et al. [The NDT (Nucleotide Divergence Threshold) method was used on each gene separately, by means of a script developed by Scarpa et al. , writtent et al. , using at et al.  genetic t et al. ).The principal coordinates analysis (PCoA) was performed using GenAlEX 6.5  on a matTripterygium delaisi, in a previous study by Koblmüller et al. [The software package Beast 1.10.4  was usedr et al. . Specifir et al. . Site par et al. . The sofr et al. . Tree Anr et al. .A total of 103 sequences for both Control Region (302 bp) and 16s gene (547 bp) were obtained in the present study. Both datasets were merged to Genbank sequences (280 for the Control Region and 136 the 16s gene) and used to implement phylogeographic analyses.Salariopsis fluviatilis  through Basic Local Alignment Search Tool (BLAST) analysis implemented in the GenBank nucleotide database (www.ncbi.nlm.nih.gov) (accessed on 24 August 2022) that showed a percentage of identity ranging from 97% to 100% for the Control Region and from 90% to 100% for the 16s gene.All the newly generated sequences were identified as belonging to the species S. fluviatilis . For the two samples from Lombardy only one haplotype was retrieved, while higher levels of divergence were found for samples from Liguria and Piedmont with 8 and 4 haplotypes, respectively.Regarding the 103 sequences obtained in the present study for the 16s gene, low levels of divergence were found for each region with a total of 7 polymorphic sites resulting in 8 haplotypes see . SamplesWith the aim to obtain a dataset including the level of polymorphism and informativeness as high as possible, these two datasets of sequences (Control Region and 16s) were concatenated to obtain a longer mitochondrial fragment, which was 849 bp long, to perform phylogeographic analyses. In this concatenated dataset, 23 polymorphic sites were found, resulting in 20 haplotypes and moderately low levels of haplotype and nucleotide diversity see . A similS. fluviatilis in Italy, the two Italian sequences (from the Lombard side of Lake Garda and Sicily) which had been previous published [The network analysis was performed on a concatenated (Control Region and 16s) dataset which included samples from northern Italy and Sardinia see  from theublished  were incGroup A included individuals from Sardinia and Liguria with the occurrence of two main star-like shapes involving the two areas. In particular, among samples from Liguria, a high level of variability was witnessed by the number of different haplotypes that are present in this area with a strong founder effect, as suggested by the occurrence of a very common central haplotype , which is surrounded by several derived haplotypes diverging for a few mutations. The haplotypes that likely originated from the lineages most common in Liguria, are private to single Ligurian individuals  or shared among Sardinian and Ligurian individuals. In only two cases, haplotypes likely derived from the Ligurian ancestor lineage are exclusive to Sardinian individuals. In particular, one of these two latter cases corresponds to the most common haplotype among Sardinian individuals, which was found in 46.8% of specimens collected and is private to Sardinia. In particular, only one haplotype isolated in the north-east of the island (Rio Mannu di Lodè river), which is exclusive to a single individual, could have not necessarily originated from the most common Ligurian and Sardinian founder lineages.Group B included samples of Piedmont and Lombardy from the present study with a marked founder effect suggested by the occurrence of a star-like shape. The two identical sequences found in Lombardy correspond to the most common haplotype occurring in Piedmont. Two out of three derived haplotypes were private of single Piedmontese individuals. The sequence from Lake Garda (Lombardy) from a previous study  was inclx-axis and y-axis and accounted for only 13.49%.In the PCoA graph see  the 61.3x-axis between two main groups (Groups A and B). Group A included individuals from Liguria and Sardinia with an internal, although poorly supported, sub-structuring. Indeed, an eastern Sardinian sample from the Rio Mannu di Lodè river .Cluster B was representative of northern Italy  and Sicily, along with the eastern part of continental Europe . Furthermore, three sequences from Spain and one from Portugal from the basin of the Guadiana River, which flows into Atlantic Ocean, set 6 to 9 point mutations apart from clusters A and B. Interestingly, a group of two Spanish and two Algerian sequences, all collected throughout a geographic area facing the Alboran Sea, sets in the network on a separate position between clusters A and B diverging for 4 point-mutations from each of them.S. cf. fluviatilis, along with sequences from Syria and Israel. Within this cluster, all the haplotypes found in Israel were exclusive to this area, while a common haplotype was shared between Turkey and Syria. Cluster C diverged from the previous reported Guadiana River group for 22 to 29 point mutations.Cluster C grouped the sequences from southern Turkey  that Belaiba et al.  identifiS. economidisi, endemic of Lake Trichonis in Greece, sets close to cluster B, 13 point-mutations apart. The two sequences representing S. atlantica, which was described in Morocco, were found 44 mutational steps far from cluster C, 50 from the Guadiana River group, and approximately 60 from clusters A and B.The sequences of S. fluviatilis together with the Mediterranean sequences taken from GenBank, and it split into a dichotomy characterized by two main clusters (clusters 1 and 2). The first cluster (cluster 1) showed a polytomic clade, including almost all S. fluviatilis sequences, and its sister clade representative of S. economidisi (represented by two sequences).The phylogenetic tree see  showed tS. fluviatilis grouped all the Italian sequences along with those from eastern Europe, mainland France, Corsica, Spain, Portugal, Switzerland, and Algeria. It includes some internal well-supported sub-clusters; among them, the three most relevant groups: (i) three Spanish and one Portuguese sequences corresponding to the Guadiana River group evidenced by network analysis, (ii) two Spanish and two Algerian sequences, and (iii) seven sequences from Crete. These three well-supported internal subgroups were also found by network analysis. The second cluster (cluster 2) of the tree included the Turkish, Syrian, and Israeli sequences, which grouped together in the cluster C of network analysis. Accordingly, with network analysis, an internal sub-structuring, consistent with the geographic distribution of samples, was also found in cluster 2 of the phylogenetic tree.The large polytomy of S. fluviatilis/S. cf. fluviatilis, the NDT and the ABGD methods converged in recognizing the same three entities. The first entity comprises Italian, French, Swiss, Algerian, Balkan and almost all Spanish sequences. The second entity includes the Turkish, Syrian and Israeli sequences corresponding to the group (including S. cf. fluviatilis) already retrieved by previous analyses. The third includes the Portuguese and Spanish sequences from the Guadiana River basin .Salariopsis included in the datasets.On the other hand, the PTP method detects four entities, partially converging with the NDT and ABGD but grouping the Guadiana River entity within the “European” entity, and splitting the group of Turkish, Syrian, and Israeli sequences into three separate entities. The GMYC methods found a unique entity for all the sequences belonging to the genus x-axis while y-axis accounted for 18.47%.In the PCoA analysis , the 41.S. fluviatilis Mediterranean populations evidenced by previous analyses, distinguishing four groups: (a) Group 1, which includes sequences from Greece, northern Italy (apart from Liguria), Sicily, Croatia, Albania, and some sequences from the western coasts of Turkey facing the Aegean and the Marmara seas; (b) Group 2, which includes sequences from Sardinia, Liguria, Spain, mainland France, Corsica, Switzerland, and Algeria; (c) Group 3, which includes the Guadiana River sequences from Spain and Portugal; and (d) Group 4, which includes sequences from Turkey that Belaiba et al. [S. cf. fluviatilis, Israel and Syria. It is important to note that group 1 and group 2 diverged only on the x-axis which accounts for only 18.47% of variation suggesting that, according to Network analysis, these two groups are structured among each other but with a weak genetic divergence.Results are consistent with the differences among a et al.  identifiSalariopsis economidisi and S. atlantica, and the two marine outgroups, S. pavo and S. basilisca, set in the plot apart from the four S. fluviatilis/S. cf. fluviatilis groups.Salariopsis species dates back to 990 kya. The common ancestor of the group of sequences, including S. fluviatilis, S. cf. fluviatilis and S. economidisi (see SEGR2_FJ465541_1 and SEGR1_FJ465540_1 in the tree) dates back to approximately 800 kya. Salariopsis atlantica (see SAMA2_FJ465526 and SAMA1_FJ465527 in the tree), which originated in 390 kya, sets in the phylogenetic tree as an external clade to the cluster including S. fluviatilis, S. cf. fluviatilis and S. economidisi.The time calibrated tree , obtaineS. fluviatilis, which includes Italian, Eastern European, French, Corsican, Spanish, Portuguese, Swiss, and Algerian individuals, along with the sequences belonging S. economidisi, differentiated approximately 610 kya. Within this group, the common ancestor to S. fluviatilis diverged approximately 575 kya, while the common ancestor of S. economidisi dates back to approximately 130 kya. Within the large clade of S. fluviatilis, it was possible to obtain a molecular dating for the three well-supported groups of sequences reported in the previous analyses. One group corresponds to the sequences from the Guadiana River basin and dates back to approximately 180 kya , another group of Spanish and Algerian sequences from the Alboran Sea area, dates back to approximately 150 kya , and the last group, including Cretan sequences, dates back to approximately 210 kya .The large group of sequences of S. cf. fluviatilis sequences, date back to approximately 630 kya.The group of sequences, including the Turkish, Syrian and Israeli The low level of genetic variation found for the 16s dataset see  may haveS. fluviatilis and two further small clusters of sequences for (1) the Guadiana River basin and (2) western Turkey (S. cf. fluviatilis) with Israel, respectively. Cluster A includes only sequences from the Italian regions of Piedmont, Lombardy, Liguria, and Sardinia, while cluster B includes sequences from Italy (only from Lake Garda in the north of the peninsula and Sicily Island), Croatia, Spain, Greece, and Turkey (in the West of the country). All the 16s sequences obtained in the present study were included within cluster A.However, even taking into account the low level of polymorphism and the reduced number of available sequences for the 16s dataset, the network analysis  was consSalariopsis fluviatilis: phylogeographic and phylogenetic analyses were performed including a high number of Italian specimens from the north-west regions of the peninsula  and the Mediterranean island of Sardinia, which represents an area so far neglected by molecular studies focused on this species. Moreover, molecular dating based on the present dataset, which covers most of the range of distribution of this species, gave us the possibility to put in an updated time range of the evolutionary patterns previously described for the new genus, Salariopsis [The present study represents the first insight into the genetic variability of the Italian populations of ariopsis ,16. Finaariopsis ,16.S. fluviatilis populations and to infer the taxonomic status of this species. In particular, the use of the mitochondrial Control Region has been effective in identifying genetically divergent groups within the species.The mitochondrial DNA showed to be a suitable tool to illustrate phylogenetic and phylogeographic relationships among S. fluviatilis populations. Indeed, the group that includes Ligurian and Sardinian samples is strongly divergent from the group formed by samples from Piedmont and individuals from Lombardy and Sicily. The genetic structuring between S. fluviatilis from Liguria and Piedmont, which do not share common haplotypes among each other, may be ascribed to the geographic isolation between these flanking geographical areas. Indeed, the genetic pattern observed here can be explained considering that the presence in the Ligurian region of boundaries, represented by the Alps ridge in continuity with the Apennines, hindered the Ligurian individuals’ colonization of the northernmost Italian regions. This scenario is known for other species of Italian freshwater fishes as well, for instance, within the genus Telestes, Stefani et al. [Squalius, the same barrier secretes S. squalus in the basins afferent to the Po River drainage network and S. lucumonis in the Tyrrhenian [Emys orbicularis) (Testudines: Emydidae) [Unio spp.  [Our research evidenced a well-supported genetic structuring among Italian i et al.  reportedrrhenian ,70,71,72mydidae)  and alsoionidae) .S. fluviatilis from Piedmont and Lombardy. This is an interesting finding, as it does not support the role ascribed to S. fluviatilis as a dispersal vector for the glochidia of the mussel of the genus Unio from Sicily to Sardinia or vice versa, suggested by Marrone and co-authors [S. fluviatilis, a higher number of individuals, from different sites in Sicily, should be analyzed. A wider sampling campaign would also allow for enlarging knowledge on the genetic distribution of this island populations.Worth noting, the sole Sicilian individual present in our dataset, from Belaiba et al. , grouped-authors . At the -authors ,76,77,78S. fluviatilis from Piedmont and Lombardy can be the result of movements of individuals from the Balkans through the Po river paleo-drainage that during the last glacial maximum included some Balkan tributaries. Indeed, the Balkan Peninsula was one of the main glacial refugia during the Pleistocene period, probably acting as a crossroad of different developmental processes [The genetic relationship that was found in the present study between samples of rocesses . FurtherS. fluviatilis samples analyzed here has been found in populations from Sardinia. As expected, this finding reflects the typical evolutionary model of species dispersal on an island. Taking as a model the migration patterns reported for the Salmo trutta lineages [S. fluviatilis may be linked to the glaciation episodes that occurred during the Quaternary. Indeed, the climatic oscillations throughout this geological event may have promoted the migration of some individuals of S. fluviatilis  from anas Plaut  demonstrS. fluviatilis during the late Pleistocene) and the Sardinian islands were renewed [Syngnathus abaster [S. fluviatilis and those of the islands [Due to low sea levels during Quaternary climatic oscillations, Sardinia may have maintained contact with the mainland via the Island of Elba, and during the Last Glacial Maximum (21 kya), connections between Corsica  genetic differentiation between two main groups of S. fluviatilis populations within the Mediterranean basin; (2) a group of populations from the Middle East represented by individuals from southern Turkey , Israel and Syria which corresponds to S. cf. fluviatilis in accordance with Belaiba et al. [Wagner et al.  and Belaa et al. ; and (3)S. fluviatilis, sequences from Italy , Greece, Croatia, Albania, and part of Turkey (western coasts areas on the Aegean and Marmara seas) are representative of the North Oriental genetic cluster, whereas sequences from Spain, Italy (with Liguria and the Sardinia Island), Algeria, France mainland, and the Corsica Island represent the Occidental genetic cluster. Interestingly, individuals from the basin of the Guadiana River, which is the only investigated freshwater basin whose tributaries flow in the Atlantic, are slightly differentiated by the Occidental genetic cluster, suggesting an incipient genetic divergence of this population from the Mediterranean clade of S. fluviatilis.Within the Mediterranean clade of S. fluviatilis too [S. fluviatilis colonized the upper Guadiana basin starting from river catches of the Júcar basin, as likely happened to the strictly freshwater fish Luciobarbus guiraonis (Cypriniformes: Cyprinidae: Barbinae) [S. fluviatilis, at least for some months [The tolerance of freshwater blennies, and ilis too , to a wiilis too . In partilis too , Almada ilis too  and Lapoilis too , it coularbinae) ,88. Nonee months .S. fluviatilis, our results also evidenced the genetic divergence of the group of a few Spanish and Algerian sequences from the area of the Alboran Sea, and the group of sequences from Crete Island, which likely originated in the interglacial period between the Mindel and the Würm glaciations.Furthermore, in accordance with Wagner et al. , within S. fluviatilis population of Crete may be regarded as a peripheral isolate , namelyS. fluviatilis, which live at the edge of the distribution of the species. The Alboran Sea can be considered a geographic area where surface marine circulation patterns may directly shape the genetic variation of marine organisms, and indirectly act on the genetic variation of brackish and freshwater species, thus producing a genetic divergence with other Mediterranean populations .S. fluviatilis sensu stricto, the geographic isolation among populations and the reduced gene flow prompted the genetic divergence among Occidental and Oriental groups of populations. In such a context, natural fragmentation or secondary contact between populations that were separated in the past but are currently present in adjacent areas may have further helped to generate the genetic differentiation observed between Liguria and Piedmont in northern Italy. However, such a divergence is not enough to be completely retrieved by species delimitation methods as a trace of incipient speciation, but it is suggestive of a relevant trend of ongoing genetic divergence.Interestingly, within the taxonomic entity Salariopsis originated approximately 1 million of years ago, before the beginning of Pleistocene glaciations. Then, repeated glacial fluctuations may have contributed to shaping the genetic divergence among fragmented populations of freshwater blennies, which were substantiated by speciation events in the genus Salariopsis. In such a context, the current distribution of S. fluviatilis and S. cf. fluviatilis lineages is the consequence of the Pleistocene post-glacial events that occurred between the end of Günz and the beginning of Mindel glaciations and that copious events of deglaciation may have led the structure of S. fluviatilis populations to be further shaped by the passage through different levels of salinity across river networks [Based on the divergence times estimated for the genetic clusters retrieved in the present study, it is possibly suggesting that the common ancestor to the Mediterranean species of networks  and Pleinetworks .S. fluviatilis to develop into a species complex as a consequence of past geological phenomena and present geographical boundaries. In this context, it is interesting to note that S. fluviatilis is separated from S. cf. fluviatilis by a number of mutations (23) that is greater than the number of mutational steps that separates it from the species S. economidisi (15), which is endemic to the western Greek Lake Trichonis [S. economidisi originated at the end of the interglacial period between the Mindel and Würm glaciations being likely contemporaneous with the Guadiana River taxonomic unit and to Alboran Sea and Crete Island genetic clusters.It is reasonable to consider the possibility that the evolutionary stages evidenced in the present study led utations 3 that isSalariopsis fluviatilis, S. economidisi and the Guadiana River taxonomic unit share a unique common ancestor that likely originated during the Günz glaciation and that is contemporaneous with the ancestor of S. cf. fluviatilis. In accordance with Perdices et al. [s et al.  and Neats et al. , these tS. cf. fluviatilis taxonomic entity could be representative of a new species, distinct from S. fluviatilis, as S. economidisi and S. atlantica, which is pending a formal morphological description and new genetic analyses. In this context, it is interesting to note that S. cf. fluviatilis shows a genetic similarity higher with the species S. atlantica, which is endemic to the Seboui river basin in Morocco [S. fluviatilis sensu strictu. This finding suggests that the Mediterranean area of North Africa could be inhabited by different freshwater species belonging to the genus Salariopsis, which may be characterized by relevant levels of genetic structuring so far undetected. What is reported here is in accordance with the phylogeographical patterns observed in other Maghrebian freshwater species , which has promoted genetic exchanges among river basins.Interestingly, the genetic divergence found for the Guadiana River taxonomic entity and also for the two divergent clusters, found within S. fluviatilis and S. cf. fluviatilis (approximately 600 kya each) and of (2) the clusters of S. economidisi, Guadiana River, Alboran Sea, and Crete Island (on average 135 kya) suggest the contemporaneous origin of these taxonomic entities. This finding supports the occurrence of similar evolutionary rates for these groups produced by the perfect timing of the mitochondrial DNA molecular clock for all Mediterranean freshwater blennies whose common ancestors likely underwent the same selective pressure prompted by similar geological conditions and geographic barriers. On the other hand, our results point out that the origin of S. atlantica is more recent in respect of the differentiation of S. fluviatilis and S. cf. fluviatilis, dating back to the interglacial period (approximately 390 kya) between the end of Mindel and the beginning of Riss glaciations.Overall, in the present study, the similar temporal estimates obtained by molecular dating for the origin of (1) Salariopsis fluviatilis sensu stricto across the Ligurian Alpine ridge and the Sardinia Island-mainland dispersal patterns, with an origin and evolutionary trajectory of founders. In particular, this research contributes to put another piece in the mosaic of the molecular data of the Italian populations and helped to understand the processes of genetic differentiation involving Mediterranean island populations whose limited effective size and high degree of fragmentation make them ideal reference models for evolutionary processes.In the present study, the hypervariable mitochondrial Control Region was shown to be highly informative in depicting the phylogeographic patterns of Italian populations of Salariopsis. Indeed, our results support the possible occurrence of a complex of species for S. fluviatilis, namely S. cf. fluviatilis  in the a et al. ,37. Indeviatilis ,10,20,25viatilis .


To analyze the correlation between susceptibility single nucleotide polymorphisms (SNPs) and the severity of clinical symptoms in children with attention deficit hyperactivity disorder (ADHD), so as to supplement the clinical significance of gene polymorphism and increase our understanding of the association between genetic mutations and ADHD phenotypes.193 children with ADHD were included in our study from February 2017 to February 2020 in the Children’s ADHD Clinic of the author’s medical institution. 23 ADHD susceptibility SNPs were selected based on the literature, and multiple polymerase chain reaction (PCR) targeted capture sequencing technology was used for gene analysis. A series of ADHD-related questionnaires were used to reflect the severity of the disease, and the correlation between the SNPs of specific sites and the severity of clinical symptoms was evaluated. R software was used to search for independent risk factors by multivariate logistic regression and the “corplot” package was used for correlation analysis.Among the 23 SNP loci of ADHD children, no mutation was detected in 6 loci, and 2 loci did not conform to Hardy-Weinberg equilibrium. Of the remaining 15 loci, there were 9 SNPs, rs2652511 (SLC6A3 locus), rs1410739 (OBI1-AS1 locus), rs3768046 (TIE1 locus), rs223508 (MANBA locus), rs2906457 (ST3GAL3 locus), rs4916723 (LINC00461 locus), rs9677504 (SPAG16 locus), rs1427829 (intron) and rs11210892 (intron), correlated with the severity of clinical symptoms of ADHD. Specifically, rs1410739 (OBI1-AS1 locus) was found to simultaneously affect conduct problems, control ability and abstract thinking ability of children with ADHD.There were 9 SNPs significantly correlated with the severity of clinical symptoms in children with ADHD, and the rs1410739 (OBI1-AS1 locus) may provide a new direction for ADHD research. Our study builds on previous susceptibility research and further investigates the impact of a single SNP on the severity of clinical symptoms of ADHD. This can help improve the diagnosis, prognosis and treatment of ADHD. Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that affects between 2 and 7% of children worldwide , 2, and Some epidemiological and clinical studies have demonstrated that genetic and environmental risk factors influence the structural and functional ability of brain networks involved in behavior and cognition in the etiology of ADHD . StudiesPrevious studies have shown that the risk of a common genetic variant of ADHD, also known as single nucleotide polymorphism (SNP) heritability, is also associated with depression, behavioral problems, schizophrenia, persistent measures of ADHD symptoms and other neurodevelopmental disorders . RecentlRecently, a combined sample of 55,374 individuals from an international collaboration for genome-wide association study (GWAS) was used to identify the first genome-wide salient loci for ADHD. This genome-wide meta-analysis provided the results of 12 ADHD-associated loci and their genome-wide significant index variations –24. SincAfter enrollment and exclusion criteria, different ADHD-related questionnaires, such as Conners parent symptom questionnaire, Stroop color-word test, Wisconsin card sorting test, et al., were used for assessment. The indicators from the questionnaires were then used to reflect the severity of different clinical symptoms of ADHD. DNA was extracted from venous blood for genotyping and bioinformatics analysis to obtain mutations in SNPs. Then the influence of SNPs on the severity of clinical symptoms of ADHD can be evaluated by correlational analysis between the indicators in the questionnaire results and the mutation status of SNPs. The workflow is shown in A total of 193 children with ADHD were included in our study from February 2017 to February 2020 in the Children’s ADHD Clinic of the author’s medical institution, including 163 males and 32 females, aged 6–15 (8.87 ± 2.17) years. Inclusion criteria: (1) meet the diagnostic criteria for ADHD according to the Diagnostic and Statistical Manual of Mental Disorders – the Fifth Edition (DSM-V); (2) complete questionnaire data; (3) complete magnetic resonance imaging (MRI) data, including sequences of T1-weighted imaging (T1WI), T2-weighted imaging and diffusion-weighted imaging (DWI), which showed no organic lesions. Exclusion criteria: (1) intellectual disability, i.e., full-scale IQ (FIQ) < 70 according to the Chinese Wechsler Intelligence Scale for Children (C-WISC); (2) serious physical diseases, such as epilepsy, congenital abnormalities, cerebrovascular diseases and autoimmune diseases; (3) other psychiatric disorders, such as autism spectrum disorders, schizophrenia, bipolar disorder, major depression, alcohol and substance use disorders; (4) drug abuse or dependence; (5) incomplete MRI data or MRI showed organic lesions; (6) family history of mental illness. The study, including all objectives and experimental procedures, has been approved by the Ethics Committee of Clinical Research in the First Affiliated Hospital of Wenzhou Medical University (Ethics Audit Number: KY2018-162).Clinical data were collected through electronic medical records. Gender, age, date of birth, grade of schooling, mode of delivery, past medical history and family history of mental illness were recorded.The C-WISC  was usedZ-score was calculated by adding up the scores and dividing it by the number of items. Each phenotype was graded based on gender and PSQ score: “Y” indicates the presence of the phenotype, and “N” indicates the opposite (The Conners parent symptom questionnaire (PSQ) was used to assess the symptoms in children with ADHD. The scale consisted of 48 items and 6 subscales, which reflect 6 clinical phenotypes of ADHD, respectively, including conduct problems, psychosomatic disorders, anxiety, learning problems, hyperactivity/impulsivity and hyperactivity indices. Each score is based on a scale of four from 0 to 3: “0” means no exception; “1” means occasionally a little or slightly; “2” means frequent or more severe; “3” means very common or very serious. A opposite , 29.The Stroop color-word test was used to assess control capabilities. The test mainly measures the ability of perceptual switching, selective attention and the ability to inhibit habitual response patterns, and is sensitive to the plasticity of mental control and response in executive function , 31. In The Wisconsin card sorting test (WCST) was used to assess the ability of abstract thinking. It consisted of 128 cards made up of different colors , shapes , and the number of shapes. The four templates were 1 red triangle, 2 pentacles, 3 yellow crosses, and 4 blue circles. In this research, the indexes used to evaluate cognitive function were: total test number, correct response number, wrong response number, persistent wrong response number, non-persistent wrong response number and completed classification number , 36.The Integrated visual and auditory continuous performance test (IVA-CPT) was used to evaluate the degree of dysfunctions in response control, attention and audiovisual integration in children. Persistent operation test software  was used to test the enrolled subjects. The IVA database automatically recorded the response control quotient, attention quotient and 29 other quotient indexes. During this study, the auditory control quotient, auditory attention quotient, visual control quotient, visual attention quotient, comprehensive control quotient and comprehensive attention quotient were selected to assess persistent attention deficits in children with ADHD –39.CAAGCAGAAGACGGCATACGAGAT and GTGACTGGAGTTCAGACGTG were added to the 5′ and 3′ ends of each probe sequence, respectively; in situ synthesis of oligonucleotides was carried out on a large scale on a chip; the oligonucleotides on the chip were eluted with ammonia water and dissolved in water to form an oligonucleotide mixture. And through PCR, forward and reverse primers (DNA was extracted from venous blood by the Phenol-Chloroform method. Multiple polymerase chain reaction (PCR) targeted capture sequencing technology was used for gene analysis. For each coding sequence of the selected gene locus, a 120 bp probe sequence was designed from the first base in the direction of 5′–3′ according to the principle of sequence reverse complementation, and there was a 60 bp overlap between each two adjacent probe sequences. Sequences of  primers  with theAfter the original Sequenced Reads were obtained, the information analysis process was carried out by referring to the genome (GRCh38/HG38), including sequencing data quality assessment and variation detection. The former mainly carries out statistics on data volume, base quality, comparison rate, coverage rate, capture rate, uniformity and other indicators, and evaluates whether the database sequencing meets the standards. If it meets the standards, subsequent analyses will be conducted. The latter, compares high-quality sequences to the human reference genome, detects the variation information in the sample, and also makes statistics and annotations of the detected variation. The multiplex PCR bioinformatics analysis process is shown in 1 was used to test the genetic balance fit degree of the Hardy–Weinberg law on population data. Correlation analysis was conducted between the indicators in the questionnaire results and the mutation of SNPs. R software (Version 4.0)2 was used to search for independent risk factors by multivariate logistic regression and the “corrplot” package was used for correlation analysis. A correlation was considered positive if the value of the correlation coefficient (rho) was >0, moderate positive if the rho was >0.2, weak negative if the rho was −0.2 ∼ 0, and moderate negative if the rho was ≤−0.2.The double-check method was adopted for data entry. SPSS statistic (Version 24.0)P > 0.05), which was representative of the population, suggesting that the population in this study was genetically balanced. However, the genotype frequency distribution of rs1222063 and rs10044618 was not consistent with the Hardy–Weinberg equilibrium test (P < 0.05), and was therefore, not representative of the population. The results are shown in Sequencing and information analysis were performed on 23 loci of ADHD children, and no mutations were detected in 6 loci . The coincidence test of Hardy–Weinberg equilibrium was performed on 17 loci with mutations. The results showed that the observations of 15 loci fit well with the expectations, conforming to Hardy–Weinberg equilibrium  and the psychosomatic disorders of PSQ had a moderate-negative correlation (the rho was −0.20), and a moderate-positive correlation with the persistent error number of WCST (the rho was 0.23); one SNP (rs223508) had a moderate-negative correlation with verbal IQ (the rho was −0.21), a moderate-negative correlation with correct numbers of WCST (the rho was −0.20), and a moderate-negative correlation with error numbers of WCST (the rho was −0.20); another SNP (rs2652511) had a moderate-positive correlation with anxiety (Grade) of PSQ (the rho was 0.24). Some SNPs, rs1427829 and rs223508, showed relatively high correlation coefficients with ADHD clinical symptoms (the rho >0.10); they had the largest number of items, 11 and 15, respectively. One SNP (rs212178) had a low correlation with the clinical symptoms and characteristics of ADHD, and there was no correlation coefficient of more than 0.10. The results are shown in P = 0.006) and FIQ  of ADHD children.As shown in P < 0.001) and anxiety (Grade) (P = 0.020), and similarly, one SNP (rs2652511) was an independent risk factor for anxiety  and anxiety (Grade) (P = 0.003). It was also found that two SNPs were independent risk factors for conduct problem ; one SNP (rs9677504) was an independent protective factor for psychosomatic disorders . The results are shown in Based on the results of the PSQ questionnaire, 5 SNPs have an impact on various clinical phenotypes of ADHD. Multivariate analysis showed that one SNP (rs1427829) was an independent risk factor for anxiety . The SNP had no effect on the reaction time of test items .Combined with the results of the Stroop color-word test and multivariate analysis, as shown in P = 0.035) or rs3768046  mutations were less likely to have an increase in the non-persistent error number, and subjects with rs1427829 mutation had an increased likelihood of persistent errors . One SNP (rs1410739) was found to be associated with the correct classification results (P = 0.049). There were two SNPs, rs11210892 and rs223508, that were found to share some similar and significant results; with those two mutations, children with ADHD were more likely to have an increase in the error number and persistent error number, and less likely to have an increase in the correct number, as shown in According to the test results of WCST, 6 SNPs had an impact on the abstract thinking ability of children with ADHD. The results of multivariate analysis showed that subjects with rs9677504  in ADHD children.The IVA-CPT is a tool to reflect the degree of children’s response control, attention and audiovisual integration dysfunction. According to the test results, we conducted a multivariate analysis, and as shown in Previous studies have confirmed that the occurrence of ADHD is affected by multiple genes. In this research, various ADHD-related questionnaires were used to reflect the severity of the disease, and the correlation between the single nucleotide polymorphism of specific sites and the severity of clinical symptoms was then analyzed. The results presented that the SNPs at different loci have an impact on the severity of clinical symptoms of ADHD, a supplement to the field of genetic research on ADHD. This indicates a role for genetic testing in the evaluation of children with ADHD. Additionally, when the same SNP shows statistical differences in several questionnaires or several sub-items of the same questionnaire, the evaluation significance of the odds ratio of different questionnaires and items is consistent, which confirms the reliability of our study.Based on the strong evidence that the dopaminergic neurotransmission system is involved in ADHD, the gene encoding dopamine transporter (DAT) on human chromosome 5  has been proposed as a candidate gene for ADHD , 26. SLCAmong the 9 loci with significantly different results in multivariate analysis, rs1410739 (OBI1-AS1 locus) affected three items from the questionnaire, including PSQ, STROOP, and WCST, respectively, corresponding to conduct problems, control ability and abstract thinking ability of children with ADHD, and all were independent risk factors. This may indicate a meaningful SNP locus that can simultaneously affect multiple ADHD symptoms was identified. Further it would provide a basis for future studies on ADHD and SNP. The SNP rs1410739  was found to be significantly associated with ADHD in the recent TWAS based on ADHD GWAS . Recent In our study, rs3768046 (TIE1 locus) was found to influence the abstract thinking ability of children with ADHD. The latest study reported that rs3768046 may change the expression of TIE1 by affecting the binding sites of transcription factors, and the expression level of TIE1 in the blood samples of patients was significantly higher than that of the control group, suggesting that TIE1 is a susceptibility gene for ADHD. The study also indicated that the G allele of ra3768046 was associated with increased susceptibility to ADHD in Chinese Han children . PreviouAnother SNP, rs223508 (MANBA locus), was found to be an independent risk factor to influence VIQ, IQ and abstract thinking ability via multivariate analysis. The protein β-mannosidase encoded by MANBA is a member of the glycosylhydrolase 2 family and acts as the final exon of β-glycosidase in the N-linked glycoprotein oligosaccharide catabolism pathway . MANBA iWe also found that rs2906457 (ST3GAL3 locus) had an effect on the severity of conduct problems in children with ADHD, and that it was its independent risk factor. ST3GAL3 is expressed in a variety of tissues, including neurons, and encodes a membrane protein  that adds sialic acid to the end of glycolipids or glycoproteins, a process that has an important impact on brain function, and St3gal3 single gene deletion mice have reduced motor coordination, cognitive impairment and behavioral hyperactivity , 71. HapThe SNP rs4916723 (LINC00461 locus) was found to affect response control, attention, and audiovisual integration. This SNP is located in the gene LINC00461, which is mainly expressed in the brain and plays a key role in the regulation of brain function –80. LINCWhat’s more, we found that rs1427829 (intron) could influence the severity of anxiety symptoms and abstract thinking ability in children with ADHD, also, rs11210892 (intron) had an effect on abstract thinking ability in children with ADHD. They have previously been found to be significantly associated with the development of ADHD in multiple replication mete-analyses , primaryIn addition, the use of “with or without” grading of PSQ projects according to the literature was verified to a certain extent. There are several limitations of our work that should be considered. First, our sample size is relatively small, so it needs to be expanded in the future in order to provide more reliable results. Second, our susceptibility SNPs were screened according to the results of previous research including GWAS, TWAS and et al., which may cause that not all ADHD-related SNPs were included in our study. Third, we focused only on children with ADHD and did not perform a comparative analysis with healthy controls. Fourth, although our research has achieved certain results, the underlying mechanism of SNPs is still unclear, which requires further cell and animal experiments to explore and verify. In the future, we plan to conduct further studies on the clinical phenotypes and neural mechanisms of ADHD in combination with more clinical data from these children.The original contributions presented in this study are publicly available. This data can be found here: NCBI: PRJNA873257.The studies involving human participants were reviewed and approved by the Ethics Committee of Clinical Research in The First Affiliated Hospital of Wenzhou Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YX and MW: conceptualization. YX, JT, AQ, XL, RZ, SC, CY, and BC: acquisition of the data for the work. YX and SL: analysis of data for the work. YX, SL, and AQ: interpretation of data for the work. YX, SL, and PV: manuscript writing. MW and AQ: funding acquisition. All authors contributed to the article and approved the submitted version.


There is growing evidence that generalised joint hypermobility (GJH) is associated with several psychiatric conditions. There are no previous studies on adult ADHD.To evaluate, in a large Swedish sample, if generalised joint hypermobility and adult ADHD are associated.431 adults with ADHD and 417 controls were included. GJH was assessed by the Beighton Score, a physical examination, and the 5PQ, a self-report screening tool. Exploratively, reported musculoskeletal symptoms and abnormal skin manifestations suggestive of symptomatic GJH (e.g. Ehlers-Danlos syndrome), were assessed to differentiate this group from the general GJH group. Logistic regressions determined the influence of an ADHD diagnosis and known covariates  on GJH and symptomatic GJH respectively.ADHD was associated to GJH, as defined by the Beighton Score and the 5PQ, with adjusted odds ratios of 4.65  and 1.86 , respectively. Likewise, ADHD and symptomatic GJH were associated with adjusted odds ratios of 6.94  and 2.66 .GJH and adult ADHD are associated conditions. Symptomatic GJH, defined as additional symptoms of pain and/or skin manifestations, has a considerably stronger link to adult ADHD than unspecific GJH has. GJH may represent a marker of an underlying systemic disorder with physical manifestations in connective tissue as well as behavioural manifestations including hyperactivity, impulsiveness and inattentiveness. Future studies should investigate if this represents a novel subtype of ADHD and if symptomatic GJH affects the ADHD management.No significant relationships.


Time-gated reflection matrix (RM) has been successfully used for optical imaging deep inside scattering media. Recently, this method was extended to enhance the spatiotemporal focusing of light ultra-deep inside scattering media. This is achieved by calibrating the decomposition of the RM with the Tikhonov regularization parameter to convert multiply scattered photons that share the same time of flight with the singly scattered photons into singly scattered photons. Such a capability suggests a reshaping to the interaction mechanism between light and scattering media, which may benefit or inspire wide optical applications that desire enhanced spatiotemporal focusing of light at depths inside scattering media. To achieve a high resolution, singly scattered (SS) photons  that only probe the in situ target need to be screened out from the predominant MS photons4. Endeavors to this purpose can be divided into two categories. In the first category, SS photons are spatially filtered out from MS photons, and the representative technology is confocal microscopy where a small pinhole aperture only allows fluorescence emission from the desired focal plane to pass through5. In the second category, SS photons are temporally filtered out from MS photons, as demonstrated in optical coherence tomography (OCT) based on time-gating technique6. For both categories, the imaging depth are still restricted to ~1 mm beneath tissue surface because the number of SS photons decreases exponentially with optical thickness7.Light is playing an increasingly important role in biomedicine from imaging, sensing, therapy, stimulation, to manipulation. The applications, however, have seen limitations at depths in biological tissue and tissue-like scattering media, where photons experience multiple scattering events due to the spatial inhomogeneities of refractive index. It is possible to exploit multiply scattered (MS) photons for imaging in some techniques, such as diffuse optical tomography8 and random matrix model in ultrasound imaging through strongly scattering media9, a time-gated reflection matrix (RM)-based method called “Smart OCT” was proposed to enhance the imaging depth11. In this implementation, a singular value decomposition of the RM was used to screen out most of the MS photons. Despite of that, MS photons with the same time of flight as the SS photons were still dominant for targets located deeper than a few scattering mean free paths (SMFP). The common wisdom is that the residual MS photons have impeded the imaging quality and hence in order to yield high resolution, they need to be removed or suppressed through methods such as iteration12 or a spatial input–output correlation11. Most recently, in Light: Science & Applications, Cao et al. suggest that the part of MS photons that share the same time of flight as the SS photons can be utilized and converted into SS photons to enhance in situ optical energy delivery spatially and temporally13.Inspired by the transmission matrix approachSS), MS2 photons (ξMS2) sharing the same time of flight as the SS photons, and other remaining MS photons (ξMS1). After the construction of RM, a singular value decomposition  is applied to the RM (R): T represents transpose, Σ is a diagonal matrix containing the real positive singular values in a descending order N is the number of the singular values in Σ), U and V are two unitary matrices whose columns correspond to the input and output mode, respectively. Singular values in the diagonal matrix corresponding to the SS, MS2, and MS1 photons are also in a descending order. Practically, Σ is not actually a standard diagonal matrix and it cannot be used to filter out the MS2 photons. Thus, matrixS is a diagonal matrix containing the square of the singular values . During the selection of λ, there is a tradeoff between the retrieval number of eigenstates and the retrieval accuracy rate of each eigenstate. In the optimization process, the target is to make the output field from calibrated matrix close to the measured output field. After successfully retrieving singular values of the SS and MS2 photons, the desired wavefront can be acquired, and the corresponding phase pattern will be loaded on the spatial light modulator. At last, the optical energy delivery can be enhanced by a magnitude at an ultra-deep (~14.4 SMFP) position.In this work, the main goal is to retrieve singular values of the part of MS photons with the same time of flight as the SS photons. First, a coherent gating is created inside the scattering medium using an ultra-short pulse beam. The back-reflected photons from the scattering medium can be divided into three types: SS photons  or the imaging depth of “Smart OCT”. Fundamentally, optical scattering arises from the interaction between photons and matter. Therefore, shaping MS photons into SS photons suggests that the light-matter interaction for the particular photons is changed and furtherly, the underlying physical mechanism can be reshaped from the conventional realm. This method may also benefit or inspire other optical applications that desire enhanced spatiotemporal focusing of light at depths inside scattering media, such as selective optogenetics


The knowledge of how the separate Attention-Deficit/Hyperactivity Disorder (ADHD) subdimensions  are associated with nonsuicidal self-injury (NSSI) and suicidal behavior (SB) is limited. The objective of this study was to investigate the associations of childhood ADHD subdimensions with NSSI and SB in children at risk of neurodevelopmental disorders .The sample (N = 391) included twin pairs where at least one twin screened positive for at least one NDD or common comorbidity at age 9 or 12. Data on ADHD subdimensions was collected through a telephone interview with a caregiver/legal guardian at age 9 or 12, and data on NSSI and SB was collected through an in-person clinical assessment at age 15. The associations between the ADHD subdimensions and NSSI or SB were tested in three different models: (1) univariable, (2) together with the other ADHD subdimensions, and (3) in a confounder-adjusted model including other NDD symptoms in addition to ADHD subdimensions, for NSSI and SB separately.A total of 32 (8.2%) adolescents reported life-time engagement of NSSI, and 18 (4.6%) SB. Childhood impulsivity was associated with SB and childhood inattention with NSSI, in all models. Hyperactivity was not meaningfully associated with any of the outcomes.Impulsivity and inattention, but not hyperactivity, may be of particular importance in understanding SB and NSSI. Brief screening for impulsivity and inattention in childhood could facilitate detection of children vulnerable to NSSI and SB and indicate valuable information for preventive and intervention strategies.The online version contains supplementary material available at 10.1186/s12888-022-04311-5. Attention-Deficit/Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder (NDD) characterized by deficient cognitive abilities that appear during childhood . WorldwiTo better understand the association between ADHD and NSSI or SB, the subdimensions of ADHD  might be of relevance. In the current diagnostic criteria of ADHD, hyperactivity and impulsivity are combined , but theThe objective of this study was to investigate the associations of childhood impulsivity, hyperactivity, and inattention with NSSI and SB in individuals at risk for NDDs (including ADHD). We hypothesized that impulsivity would be positively associated with both NSSI and SB. Based on the conflicting prior knowledge, potential difference in strength of association between impulsivity and NSSI relative to SB, was investigated with an explorative approach. Furthermore, considering the conflicting and limited prior knowledge regarding inattention and hyperactivity, respectively, these subdimensions were investigated in a more exploratory manner.strengthening the reporting of observational studies in epidemiology checklist (STROBE)  and screen-negative [N = 199]), the results were uncertain with statistically non-significant results in almost all models. Hence, the generalizability of the main results to any of the subgroups specifically could not be confirmed. Results are found in Table S6 and S7.The objective of this longitudinal study, following children at risk for NDDs into adolescence, was to investigate the associations of childhood impulsivity, hyperactivity, and inattention with NSSI and SB. The results showed that childhood impulsivity was associated with SB, childhood hyperactivity was not associated with either NSSI or SB, and lastly, that childhood inattention was associated with NSSI.In accordance with our expectations, and results from previous meta-analyses –31, impuHyperactivity was not associated with either NSSI or SB in this study. These results indicate that impulsivity seems to be the driving factor in the association between impulsivity and hyperactivity combined, and NSSI or SB. This result points at the need to study and consider these ADHD subdimensions separately.In line with one previous study  and contIn summary, the findings indicate that the associations between the separate ADHD subdimensions and NSSI and SB may vary. The findings inform about opportunities to detect information in childhood associated with self-injurious behaviors with average onset in adolescence. These findings could be clinically meaningful as children at risk of NDDs might be presented to health care where impulsivity and inattention could be screened for and valuable independent of what the child is referred for. To prevent and intervene on NSSI and SB, interventions aiming to increase impulse control and attentional ability could be valuable.Although this study has several strengths, such as: population-based recruitment, including both boys and girls, and semi-structured clinician-assessed outcome measures with the possibility to distinguish NSSI from SB, there are also some limitations to highlight. First, this study was constrained by statistical power, where some of the associations might not have been confirmed for this reason. Regarding the prevalence of NSSI and SB, the prevalence of SB was similar to previous findings, but the prevalence of NSSI was lower compared to community samples of adolescents up to age 18 . HoweverGiven the novelty of some of the results, and the few studies on the associations between ADHD subdimensions and NSSI and SB, replications are needed. Further research could also extend the knowledge by looking closer at the effects of ADHD subdimensions on NSSI and SB separated by gender, different frequency, and severity levels of NSSI and SB, and the effect on NSSI and SB alone as well as the combination of NSSI and SB.In this longitudinal study following children at risk for NDDs over the transition to adolescence, the findings were that: (1) childhood impulsivity was associated with SB, (2) childhood hyperactivity was not associated with either NSSI or SB, and (3) childhood inattention was associated with NSSI. Brief screening of inattention and impulsivity could offer opportunities to detect children vulnerable to NSSI and SB, and these symptoms could inform preventive and intervention strategies.Below is the link to the electronic supplementary material.Supplementary Material 1. Supportive information