The inhibitory strength of these chemicals on rat 11β-HSD1 was much weaker than that on the human enzyme, ranging from 74.22 to 205.7 µM. All BPA analogues are mixed/competitive inhibitors of both human and rat enzymes. Molecular docking studies predict that bisphenol H and bisphenol G both bind to the active site of human 11β-HSD1, forming a hydrogen bond with catalytic residue Ser170. The bivariate correlation of IC50 values with LogP (lipophilicity), molecular weight, heavy atoms, and molecular volume revealed a significant inverse regression and the correlation of IC50 values with ΔG (low binding energy) revealed a positive regression. In conclusion, the lipophilicity, molecular weight, heavy atoms, molecular volume, and binding affinity of a BPA analogue determine the inhibitory strength of human and rat 11β-HSD isoforms.Bisphenol A (BPA) analogues substituted on the benzene ring are widely used in a variety of industrial and consumer materials. However, their effects on the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) remain unclear. The inhibitory effects of 6 BPA analogues on the inhibition of human and rat 11β-HSD1 were investigated. The potencies of inhibition on human 11β-HSD1 were bisphenol H (IC Bisphenols are a group of chemical compounds that are widely used in the production of plastics, resins, and other materials. Among the most commonly used bisphenols is bisphenol A (BPA). Bisphenols have been in use for decades and can be found in a wide range of products, including food packaging, water bottles, medical devices, and electronics .However, concerns about the potential health effects of bisphenols have led to increased scrutiny of their use. Bisphenols have been identified as endocrine-disrupting chemicals (EDCs), meaning that they have the ability to interfere with the body’s hormonal systems ,2,3,4. TOne of the most well-known bisphenols is BPA, which has been extensively studied for its potential health effects. BPA has been linked to a variety of health problems, including breast and prostate cancer, obesity, and fertility issues ,2,3,4.However, due to its potential harmful effects on human health, regulators have been increasingly concerned about the use and exposure of BPA ,5,6. StuDespite these bans and restrictions, BPA continues to be used in many consumer products. In addition, BPA exposure can occur not only via ingestion from food and beverage containers but also via dermal contact and inhalation ,3,7.However, as awareness of the potential health risks associated with BPA has grown, manufacturers have begun to replace it with other BPA analogs, such as 4,4′- bis (methoxy benzene) (BPAME) and 2,2-bis(2-hydroxy-5-biphenylyl) propane . While these analogs were initially thought to be safer alternatives to BPA, recent research has raised concerns about their potential health effects as well.According to ECHA, there are over 60 BPA analogues, including one group of potential alternatives to BPA with substitutions on the benzene ring. These compounds contain either alkyl groups, such as methyl , propyl bisphenol A , or allyl , or aryl groups, such as aryl bisphenol A (BPH) ,9,10. BPOne potential target of BPA is its effects on glucocorticoid metabolism. Glucocorticoids, such as cortisol (F) in humans and corticosterone (CORT) in rats, are corticosteroid hormones that bind to the glucocorticoid receptor, a nuclear receptor that regulates gene expression ,14,15. GThe 11β-HSD1 enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family, with the catalytic motif Ser/Thr…Tyr-X-X-X-Lys residues . For hum50 values of BPA and BPA analogues ranged from 0.78 to 319.16 µM  was adopted as the target for docking cortisone. Cortisone forms two hydrogen bonds with catalytic residues Ser170 (3.28Å) and Tyr183 (3.03Å) A,B. Firshttps://saves.mbi.ucla.edu/, (accessed on 11 May 2023)). It was found that 100 percent of residues fell inside the favored (94.6 percent) and the additionally allowed (5.4 percent) regions, whereas no residues fell within the disallowed region for rat 11β-HSD1  was adopted as the target for molecular docking analysis. The structure was vilified by Ramachandran plot analysis ProCheck software in the UCLA-DOE LAB-SAVES v6.0  when compared to BPA. This inhibition is possibly due to an increase in lipophobicity. It is true that the benzyl substitutions on the benzene ring, as in BPH, significantly increase the LogP value. Although BPG and DABPA have the same carbon number, the double bond allyl group in DABPA causes the reduction in LogP, thereby leading to a weaker inhibition than BPG. Interestingly, the ether in BPAME significantly increases the LogP (4.04 in BPAME vs. 3.42 in BPA), thereby increasing the inhibitory strength when compared to BPA.The study also noted that TMBPA was weaker than DMBPA to inhibit human and rat 11β-HSD1 enzymes, despite having an extra methyl group, which would generally increase lipophobicity (LogP: 4.66 in TMBPA vs. 4.04 in DMBPA). The reasoning is that the additional methyl group may increase the space hindrance. This essentially means that the extra group creates more spatial challenges for the molecule to interact with its target, which could result in less activity.In this study, unlike human 11β-HSD1, we found that BPA analogues are much weaker in their ability to inhibit rat 11β-HSD1 when compared to BPA. For example, when the IC50 values are compared, BPH is about 99 times more potent to inhibit human 11β-HSD1 than rat 11β-HSD1, and BPG is about 21 times more potent to inhibit the human enzyme than the rat enzyme . AlthougThe human and rat 11β-HSD1 enzymes belong to the SDR family, which has catalytic residues Ser…Tyr-X-X-X-Lys . DockingAlthough the crystal structure of human 11β-HSD1 is available, the structure of rat 11B-HSD1 remains unavailable. This presents a challenge for studying the effects of BPA analogues on this enzyme in rats. However, after analyzing the differences and similarities between rat and human 11β-HSD1, we noted a subtle difference between the two: the binding pocket size of human 11β-HSD1 is larger than that of the rat enzyme . It rema50. This suggests that compounds with stronger binding affinity to 11β-HSD1 are also more effective inhibitors.In addition, it has been found that binding energy, or the energy required for the compound to form a stable complex with the enzyme, is positively correlated with IC50 value, while others are much weaker, indicating that other BPA analogues except BPH might be safe. It is emphasized that care should be taken when extrapolating results obtained from the rat model to humans.It is important to note that the use of BPA analogues does not necessarily mean that they are a safe alternative to BPA. This study found that the inhibitory strength of BPA analogues was BPH > BPG > DABPA > DMBPA > BPAME > TMBPA = BPA, indicating that they are more potent to induce dysfunction due to cortisol deficiency. However, only BPH has a sub-micromolar ICThe inhibition of 11β-HSD1 may lead to adverse effects. Cortisol is an important hormone involved in the body’s stress response, and inhibition of 11β-HSD1 can result in cortisol deficiency. This can lead to adverse effects such as fetal lung development disorders, excessive ACTH stimulation, and liver fibrosis.BPA analogues also target other steroidogenic enzymes. For example, BPA or BPA analogues can inhibit human and rat 11β-HSD2  ,24, humaWe also predicted that the aqueous solubility level was BPA > BPAME = BPG = DABPA = DMBPA= TMBPA > BPH, and they were found to be able to transport across the BBB and inhibit the drug metabolic enzyme CYP2D6, except TMBPA. In addition, BPH and BPG had lower absorption levels in the human intestine and all had hepatotoxic potential. These results suggest that these chemicals might target the tissues to inhibit 11β-HSD1.11-dehydrocorticosterone (DHC), corticosterone (CORT), cortisone, and cortisol were obtained from Steraloids , BPA , dimethyl sulfoxide (DMSO), G6P, and NADPH from Sigma-Aldrich , 4,4′-bis(2-isopropylphenol) , 4,4′-bis(methoxybenzene)  and 2,2′-diallyl BPA  from Rhawn , 2,2-bis(2-hydroxy-5-biphenylyl)propane  and 2,2-bis(4-hydroxy-3-methylphenyl)propane  from TCI , and 2,2-bispropane  from JK Chemical . The human 11β-HSD1 enzyme in the human liver microsome (CAT# H2610) was obtained from Iphase . The rat 11β-HSD1 enzyme in the rat liver microsome was prepared from livers of male Sprague-Dawley rats (age 3 months) from Shanghai Laboratory Animal Center  under the approval of the Wenzhou Medical University Institutional Animal Care and Use Committee (wydw2020-0801).g for 30 min, at 14,500× g for 30 min, and at 105,000× g for 60 min twice to produce the microsomal pellet. The pellets were resuspended in cold PBS by homogenization, and the concentration of microsomal protein was determined using the Bio-Rad Protein Assay Kit  according to the manufacturer’s instructions.The preparation of rat liver microsomes was carried out as previously reported . In this50) assay: 200 nM cortisone or DHC and 0.1, 1, 5, 10, 25, 50, or 100 µM BPA analogues in the system for 15 min; (5) Mode of action assay: 0, 0.25, 0.5, 1, 2, 3, and 4 µM cortisone or DHC, and 0–50 µM BPA analogues in the system for 15 min. At the end of the reaction, acetonitrile  and internal standard testosterone-d5  were added to the system, the organic layer was extracted, and 10 µL was injected into the HPLC-MS/MS system to measure cortisol, or CORT, as previously described /(KmApp + [substrate]), where VmaxApp is the adjusted Vmax and KmApp is the adjusted Km, VmaxApp = Vmax/(1 + [BPA derivative]/(α ∗ Ki), where [BPA derivative] is the inhibitor concentration, and KmApp = Km ∗ (1 + [BPA derivative]/Ki)/(1 + [BPA derivative]/(α ∗ Ki)), where Ki is the inhibition constant. “α” was used to judge the mode of action: when α is very large, the BPA derivative is a competitive inhibitor; when α is very small but greater than zero, the BPA derivative is an uncompetitive inhibitor; when α is 1, the BPA derivative is a noncompetitive inhibitor; when α > 1 or α < 1, the BPA derivative is a mixed inhibitor. The lineweaver-Burk plot after linear regression was also carried out to judge the mode of action.The time curve of the 11β-HSD1 reaction was prepared by a linear regression using GraphPad version 8.0 . A nonlinear regression fit (curve fit) using the Michaelis-Menten equation (V = Vmax ∗ [substrate]/(Km + [substrate]), where V is 11β-HSD1velocity (pmol/mg/min), Vmax is maximal velocity, [substrate] is substrate concentration, and Km is the Michaelis-Menten constant) was performed by GraphPad, and Km and Vmax were calculated. Inhibitory effects of BPA derivatives at 100 μM were compared to the control . Dose-response was performed using a range of concentrations of BPA derivatives, and a nonlinear regression (curve fit) of Log[BPA analog] vs. response (three parameters) was carried out by GraphPad to calculate the half-maximal inhibitory concentration  with a synthetic inhibitor and NADP targets . AlphaFo targets  was sele targets ,32. The  targets ,34. ProC targets . Docking targets , and con targets . After d targets . After d targets .3, ring number, and molecular weight (MW), were obtained from the ZINC database [50 values of each BPA analogue was analyzed by Pearson correlation analysis.Structural features, including hydrogen bond donor, hydrogen bond acceptor, heavy atoms, hetero atoms, LogP, fraction spdatabase . ΔG was In this study, we used Discovery Studio software v19.1.0  to predict the drug metabolism and pharmacokinetics (ADMET) parameters of seven bisphenols. Physical descriptors such as aqueous solubility, blood–brain barrier (BBB) penetration, human intestinal absorption (HIA), hepatotoxicity, cytochrome P450 2D6 (CYP2D6) inhibition, and plasma protein binding (PPB) were generated from the analysis .50 values between each BPA analog was judged by one-way ANOVA followed by a post hoc Turkey’s multiple comparisons test. Data were presented as means ± SEM, and differences were statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.The experiments were repeated four to six times. The inhibitory potency of BPA analogues at 100 µM was compared by a one-way ANOVA, followed by a post hoc Dunnett’s multiple comparisons test to judge any significant difference between BPA analogues and the control. The significant difference in ICIn conclusion, in this study, we found that 11β-HSD1 is a new potential target of BPA analogues. BPA analogues with substitutions on the benzene ring significantly increase the binding to human 11β-HSD1, thereby leading to more potent inhibition of the enzyme activity. The inhibitory strength of BPA analogues was BPH > BPG > DABPA > DMBPA > BPAME > TMBPA = BPA, indicating that they are more potent to induce dysfunction due to cortisol deficiency. However, there is species-dependent inhibition with the rat enzyme, which is insensitive to BPA analogues. Hydrophobicity seems to determine the inhibitory strength of BPA analogues on both enzymes. Docking analysis reveals the binding potency of BPA analogues to 11β-HSD1 might depend on lipophilicity, molecular weight, molecular volume, and heavy atoms.


Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays.Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic 50 values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 103 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH.IC50 values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC50 of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification.The IC50 values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system.Results from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody ICThe online version contains supplementary material available at 10.1186/s12936-023-04765-2. Anopheles spp. mosquito during a blood meal, releasing sporozoites into the dermis and initiating an infection ) values were calculated from the ROI and used to determine relative infectivity.Liver-stage luciferase activity was visualized using an intensified-charge-coupled device (I-CCD) video camera of the IVIS. Mice were anaesthetized using isoflurane anesthesia system . Following this, mice were injected intraperitoneally (IP) with l-Glutamine (Gibco) and 40 µg/ml Gentamicin (Sigma-Aldrich) at 37 °C (5% CO2). At ~ 90% confluency, cells were split 1:6 into a pre-coated collagen flask (Corning™) every 3–4 days. The cell line was authenticated via American Type Culture Collection Human STR Profiling Service (ATCC) and tested negative for mycoplasma contamination (Fisher Scientific).Hepatoma cells (HC-04) were maintained in Minimum Essential Media (Gibco) and Ham’s F12 nutrient mix (Gibco), supplemented with 30 mM HEPES (Gibco), 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 2 mM 2 incubator. The mAbs were diluted  in 6% BSA in RPMI-1640 medium (Corning) in 80 μl and incubated at 37 °C in a CO2 incubator. After salivary gland dissection and sporozoite preparation, 80 μl of sporozoites (150 sporozoites/µl) was added to each mAb dilution and control and 40 μl was dispensed to each well in triplicates. Plates were centrifuged at 200×g for 3 min and imaged using Cell Insight CX7 high-content imaging system (Thermo Fisher Scientific) in brightfield. Wells were imaged at 20X at the rate of one frame/second for 60 s. Acquired images were exported for analysis and sporozoites were manually quantified as gliding and non-gliding. Gliding was defined as attached movement, and percent gliding was calculated by dividing total gliding sporozoites by total sporozoites.The live gliding motility assay was performed as previously described with minor changes . Briefly3 sporozoites from each treatment were added to wells with HC-04 cells in triplicates. The plate was centrifuged at 200×g for 5 min, and sporozoites were allowed to invade HC-04 cells for 1 h at 37 °C in CO2 incubator. After one hour, the plates were washed with media to remove uninvaded sporozoites and a subsequent media change at 24 hpi. At 48 hpi, infected HC-04 wells were incubated with 1 × Xenolight (PerkinElmer) for 10 min at 37 °C. The plates were imaged on the CLARIOstarPlus (BMG LABTECH) immediately after Xenolight incubation for relative luminescence units (RLU) output.ILSDA was carried out as previously described with minor changes . HC-04 cP. berghei 18S rRNA (PBANKA_0521221) normalized to murine GAPDH  (NM_001289726.2) copy number as previously described [RNA was isolated from Pb-PfCSP and PbmCh-luc infected liver samples for qRT-PCR. Infected liver samples were homogenized in TRIzol (Fisher Scientific) on a TissueLyser II (Qiagen). RNA was coprecipitated with glycogen (Sigma-Aldrich) at − 20 °C overnight and treated with DNase (Fisher Scientific). cDNA was synthesized using SuperScript III Platinum two-step quantitative reverse transcription-PCR (qRT-PCR) kit (Invitrogen), according to manufacturer’s instructions. The LS burden was quantified by absolute copy number of escribed .8 through 101. cDNA samples were quantified by Luna® Universal qPCR Master Mix (New England BioLabs) along with the standards and copy numbers were used to determine the gene expression.Sporozoite surface protein essential for liver stage development  and liver-specific protein 2  served as early and late LS markers, respectively , and data were analysed using GraphPad Prism version 9 . % Inhibition was calculated by the equation below:Significance between means was determined by a two-way ANOVA followed by a Tukey’s multiple comparisons post hoc test with relevant comparisons for sporozoite gliding assay. Where indicated, a Kruskal–Wallis test with Dunn’s multiple comparisons and a two-tailed Mann–Whitney test were used for analysing non-parametric data. ICP. berghei sporozoites in a live gliding assay was evaluated first  and 2D10 (anti-PfCSP) inhibited sporozoite gliding in a dose-dependent manner and were not cross-reactive as PbmCh-luc exposed to 2A10 and Pb-PfCSP(r) exposed to 3D11 exhibited gliding motility similar to control  sporozoites respectively. Complete inhibition of PbmCh-luc and Pb-PfCSP(r) live sporozoite gliding was observed at a concentration of 250 µg/ml for mAbs 3D11 and 2A10, respectively  than 3D11 against PbmCh-luc  was observed of Pb-PfCSP(r) LS forms  infected mice  received sporozoite challenge infections. Upon challenge with transgenic own Fig. a and d. own Fig. b and e wown Fig. c and f. ice Fig. f. Howeveice Fig. c.Fig. 3PLastly, since a dose-dependent antibody reduction of both parasite lines were observed at equivalent concentrations in vivo, the relationship between functional inhibition in vitro to in vivo was explored further Fig. . AdditioPbmCh-luc [Pb-PfCSP(r) [P. berghei and P. falciparum CSP, respectively [D of 2.7 ± 2.1 nM [P. falciparum CSP while 3D11 mAbs have a high affinity,KD of 159 ± 47 nM [P. berghei CSP [Functional assays replicate critical invasive sporozoite phenotypes in vitro , 55, 60.bmCh-luc  and Pb-PPfCSP(r) , 62) andectively , 63, 64.± 2.1 nM , to ‘NAN50 concentrations were determined against in vitro liver-stage development using HC-04 cells. The observed disparity between IC50 values of 2A10 and 3D11, despite antibodies exhibiting comparable inhibition at the highest concentrations tested, may be related to differences in epitope binding affinity and avidity which was not explored in this study  , with an observed IC50 of 0.4 µg/ml [PfCSP@PbUIS4 [PbmCh-luc  as the lower concentrations assessed in vitro had less than 80% inhibitory. Previous studies required a higher dose of 2A10 to reduce liver burden compared to the in vivo data from this study [P. berghei liver burden was reduced by 75% to 82% in C57BL/6 mice that received 300 µg of 2A10 [6P. falciparum sporozoites [Following determination of the ICis study , 63, 67.rozoites . In a morozoites , a signiPbmCh-luc in vitro and in vivo was lower than between 2A10 inhibition of Pb-PfCSP(r) in vitro and in vivo (Fig. , and comparing inhibition in vitro across primary cells, immortal cell lines and other rodent models [In the current study, the difference between 3D11 inhibition of ivo Fig. . These ft models , 73. ThiP. berghei LS forms in in vitro and in vivo assays. While 2A10 mAbs had higher inhibitory activity in vitro and in vivo than 3D11 mAbs, the discrepancy in inhibition between both assays was more pronounced for 2A10 than 3D11. Despite some similarities between antibody inhibition titration curves, no significant correlation was established in parasite burden between these assays by luminescence. While not optimal for predicting in vivo outcomes, in vitro assays remain a useful preliminary screening tool as high throughput cost-effective laboratory assays for vaccine discovery.This study evaluated antibody-mediated inhibition of transgenic Additional file 1: Figure S1. Gene expression levels of early and late liver-stage P. berghei biomarkers. Figure S2. Correlation between relative luminescence and P. berghei 18S rRNA expression in infected mice. Table S1. Primers used in qRT-PCR for LS quantification and maturity biomarkers.


Biomolecular condensate is an emerging structural entitythat regulatesvarious cellular processes. Recent studies have discovered the phase-separation(PS) capability of several transcription factors (TFs) including YAP/TAZupon biological stimuli, which provide new mechanisms of gene regulation.However, it remains mostly unanswered as to whether PS from a diffusestate to a phase-separated state promotes gene transcription. To addressthis question, we have designed a chemogenetic tool, dubbed SPARK-ON,which manipulates the PS of YAP and TAZ without a biological stimulus,forming condensates that are transcriptionally active, containingthe DNA-binding partner TEAD, genomic DNA, transcriptional machinery,and nascent RNA. Most importantly, PS of TAZ increases the transcriptionof its target genes. Therefore, our data indicate that PS promotesgene transcription of TAZ. SPARK-ON is advantageous to current mutagenesis-basedapproaches that are often problematic when mutagenesis affects thetranscriptional activity of a TF. Furthermore, protein abundance levelsalso affect gene transcription, but PS depends on protein abundancebecause PS occurs only when the protein level is above a saturationconcentration. SPARK-ON decouples PS from protein abundance levelswithout introducing mutations and thus will find important applicationsin understanding the biological roles of PS for many TFs and otherbiomolecular condensates. Chemogenetictool SPARK-ON decouples phase separation fromprotein levels, drives transcription factor condensate formation,and enables dissection of the biological role of phase separation. Two major breakthroughsin the fundamental understanding of these structures have revealedthat biocondensates  form via phase separation (PS).2 PS is often driven by multivalent interactions.3 Multivalence is introduced by folded multidomainsin a protein or single folded domains that form oligomeric complexesas well as by intrinsically disordered regions (IDRs) that containmultiple charged or aromatic residues mediating weak and multivalentinteractions or via the stickers-and-spacers model.4 Functional studies indicate that many condensates are biologicallyactive such as in cell signaling and gene transcription.10 For example, recent studies of several TFs show that they undergophase separation (PS) and form biomolecular condensates,17 including transcriptional effectors of the Hippo pathway, YAP andTAZ, when the concentration of the proteins surpasses a thresholdcaused by an environmental stimulus such as osmotic stress for YAP.18 These TF condensates are further shown to betranscriptionally active.9 For example, the YAP and TAZ condensates contain transcriptionalmachinery.20Biomolecular condensates are importantbiological structures thatplay key roles in multiple cellular processes.17 whether phase separation reallychanges the transcriptional output is still controversial.21 Answers to this key question are hampered byconceptual and technical challenges. Several studies introduced mutationsto change the phase behavior in order to correlate the driving forcefor phase separation with transcription.18 These mutations are often introduced into the TF’s activationdomain that harbors the PS-promoting IDRs, but the activation domainsoften interact with the mediator that loops enhancer to promoter viainteraction with RNA polymerase II and general transcription factors.22 Thus, the mutations that are introduced intothe activation domain in order to block PS will likely also impactthe ability of TFs to form diffuse complexes with transcriptionalmachinery.23 Unfortunately, many previousstudies reached conclusions that phase separation-activated transcriptionis based on the PS-blocking mutations that reduce transcription withoutevidence that the introduced mutations do not affect the interactionof the TFs with the mediator and transcriptional machinery.23 This suggests that the reduction in transcriptional output can bedue to the blocked PS and/or the reduced interaction with the transcriptionalmachinery. Therefore, it is still under debate if the transcriptionaloutput in the presence of TF condensates would also be achieved inthe absence of phase separation by diffuse complexes. Determiningthe role of phase separation in transcription is thus much needed.The conceptual and technical challenges call for new tools that enableus to assess transcriptional activity upon dissolving condensateswithout introducing mutations or changing expression levels.Although many TF condensates contain transcriptionalmachinery,62 and chemogenetic tools such as the FKBP/Frb-based systems as wellas the eDHFR/HaloTag-based tools.64 Such tools enable us to decouple the role of phase separation fromthe change in protein expression levels. Optogenetic tools achievethe subcellular manipulation of protein phase separation and are veryvaluable in understanding biological condensates.56 On the other hand, one advantage of chemogenetic tools is that asmall-molecule-based approach simplifies sample processing with alarge number of cells for biochemical characterization including geneticanalysis such as RT-qPCR. While chemogenetic tools such as the rapamycin-inducibleFKBP-Frb system have been used in manipulating PS,57 they have not been applied to the phase separation of transcriptionfactors, and the rapamycin-mediated FKBP-Frb interaction is strongerthan those in the biomolecular condensates that mostly form via weakinteractions.Enabling technologies, that are capable of manipulating PS withoutchanging protein levels or introducing mutations, can help us gaina mechanistic understanding and appreciation of the functional rolesof phase separation. These technologies include optogenetic toolssuch as the Cry2-based OptoDroplet and the ferritin/iLID/sspB-basedCoreletHere, we decided to engineer a new chemogenetictool that is capableof manipulating PS and contains several unique capabilities: (1) ituses FDA-approved drug molecules; (2) the PPI pair is smaller thanthe FKBP-Frb pair, with one component as small as ∼30-aa; (3)the interaction is weak so that it better mimics the weak interactionin most of the biomolecular condensates; and (4) the drug-inducedweak interaction allows the quantitative manipulation of PS and thusenables us to determine role of PS in a quantitative manner. Suchversatile tools enable us to decouple the role of phase separationfrom a protein expression level-induced or mutation-induced changein protein activity. Furthermore, small molecule-activatable chemogenetictools that are compatible with genomic analysis approaches will facilitatefunctional characterizations such as the profiling transcription ofspecific genes. Such chemogenetic tools are powerful technologiesin determining the role of phase separation in gene regulation.de novo designed multivalent tags into the heterodimer toinduce multivalent PPIs that drive PS. This small molecule-activatablechemogenetic tool is capable of driving PS and forming liquid-likecondensates that are biologically active. The engineered chemogenetictools are compatible with genetic analysis approaches including RT-qPCR.To engineer such versatile chemogenetic tools, we first designeda small molecule-inducible protein heterodimer. Then we introduced 24 IMiDs, including thalidomideand lenalidomide, are FDA-approved drugs against multiple myelomaand have no or little toxicity in other cells.25 IMiD-dependent interactions between CRBN and IKZF1 bringthe transcription factor IKZF1 to the cullin ring E3 ubiquitin ligasecomplex CUL4-RBX1-DDB1-CRBN via the interaction between CRBN and theadaptor protein DDB1, resulting in ubiquitination and degradationof IKZF1.26 To design a stable IMiD-controllableprotein heterodimer, we sought to disrupt the interaction betweenCRBN and DDB1. Structural studies of DDB1-CRBN-IKZF1 show that thehelical bundle domain (HBD) of CRBN interacts with DDB1 but does notbind lenalidomide or interact with IKZF1  of CRBN binds lenalidomide and interactswith IKZF1, without direct contact with DDB1. Additionally, the N-terminaldomain (NTD) of CRBN does not appear to interact directly with DDB1or IKZF1. Therefore, we first truncated the NTD and HBD of CRBN butretained the 109 amino acid (aa) CBRN CTD, which was renamed CEL.Next, we truncated IKZF1 so that only the zinc finger 2 (ZF2) domainwas retained because ZF2 binds lenalidomide and interacts with CEL-inducible PPI pair of cereblon (CRBN) and Ikaros (IKZF1)because our previous study showed that IMiDs such as lenalidomidecan induce condensate formation when the protein pair is tagged withmultivalent tags.th IKZF1 1A.27 On with CEL1A.27 Theo as ZIF 1A. We veo as ZIF 1B.Movie S1). SOScat continued to accumulateat PM at later time points, and membrane ruffling became visible (Movie S1). We also confirmed that the total proteinlevel of SOScat did not change during this process upon addition oflenalidomide . Todetermine whether SOScat translocation activates ERK, we used a GFPtranslocation-based ERK activity reporter called ERK-KTR, which istranslocated from the nucleus to the cytoplasm upon activation ofendogenous ERK.28 Upon addition of lenalidomide,ERK-KTR translocated from the nucleus to the cytoplasm .29 Thus, we have demonstrated that the lenalidomide-inducibleCEL and ZIF heterodimer can be used to control PPI and cell signaling.To demonstrate that the engineered CEL and ZIFcan be used to controlPPI via lenalidomide, we tested whether this system can drive subcellulartranslocation of the GTP exchange factor SOS to the plasma membrane(PM). The presence of SOS on the PM can activate Ras and promote ERKactivity via the MAPK pathway 1C, left.3 We fusedCEL and ZIF to the homo-oligomeric tags (HOTag) that are denovo designed coiled coils: CEL to HOTag3 (30 aa) and ZIFto HOTag6 (33 aa) . HOTag3and HOTag6 have previously been characterized as a hexamer and tetramer,respectively.31 To visualize phase separation, we tagged both constructswith the enhanced GFP (EGFP) (Movie S5). We named this technology SPARK-ON . For proteins that can form condensates,they often contain a multivalent domain or an IDR. The SPARK-ON chemogenetictool is engineered to drive the PS of such a protein of interest (POI)by lenalidomide-inducible multivalent interactions. SPARK-ON-taggedPOI is referred to as POI/SPARK-ON, which is composed of two constructs:CEL-EGFP-POI and ZIF-EGFP-HOTag6. In the above demonstrated case,POI is HOTag3. As shown below, POI can also be a protein that hasa tendency to form condensates, such as a TF .To engineer the CEL/ZIF system into a chemogenetictool that can control protein PS, we introduced multivalency intothe lenalidomide-dependent PPI system because multivalent PPI candrive protein phase separation.We quantified the time-lapse images by calculatingthe SPARK signal,which is defined as the ratio of the summarized pixel intensity ofdroplets divided by the summary of the pixel intensity of the totalcellular fluorescence 2C. QuantMovie S6). The fusing droplets initially formed a dumbbell shape,which over time relaxed to a spherical shape  to be 3.07 ± 0.46 (s/μm) . The disassemblyprocess was quantified by calculating the SPARK signal. The reversibilityof these HOTag3/SPARK-ON droplets is consistent with the above characterizationthat they are liquid droplets. Solid-like condensates are known tobe irreversible. Finally, the titration of lenalidomide in the cellsshowed that the degree of droplet formation  was dependent on the concentration of lenalidomide with ahalf-to-maximum value of ∼0.3 μM  would recruitclient proteins and whether the phase separation of a client proteinwould recruit scaffold proteins. We applied the SPARK-ON technologyto proteins of stress granules (SG). Recent studies have revealedthat G3BP1 plays a central role in SG assembly,Methods). First, we preformed SparkDrop-Frb by incubatingthe transfected cells with lenalidomide (Movie S8). Furthermore, red fluorescent FUS droplets were observed afterthe formation of G3BP1 droplets, which colocalized with both near-infraredand green droplets (Movie S8). And the totalprotein levels of G3BP1 and FUS underwent no or little change duringthese procedures .In control experiments, rapamycin alone (lack of FKBP or Frb) didnot induce G3BP1 phase separation or subsequent FUS recruitment . Thus, these imaging studiesindicate that the phase separation of SG scaffold protein G3BP1 recruitsthe SG client protein FUS.We first determined whether SPARK-ON-inducedG3BP1 condensatescould recruit FUS and TIA-1 in living cells (without applying a stressstimulus such as arsenite), and then we examined whether FUS or TIA-1phase separation could recruit G3BP1. We combined a lenalidomide-inducibleSPARK-ON droplet (SparkDrop) with the rapamycin-inducible FKBP andFrb heterodimer. We incorporated Frb into SparkDrop (referred to asSparkDrop-Frb), fused full-length G3BP1 to FKBP tagged with IFP2,and also tagged FUS with a red fluorescent protein mKO3 . In control experiments,rapamycin alone (lack of FKBP or Frb) did not induce FUS phase separation. Thus, the imagingstudies show that phase separation of the SG client protein FUS doesnot recruit the SG scaffold protein G3BP1.To induce phase separation of the client proteinFUS and determinewhether G3BP1 is recruited, we fused FKBP to FUS tagged with mKO3and fused G3BP1 to IFP2 3D. We prSupporting Figure S8A–C), and rapamycin alone (without FKBP or Frb) does not recruit TIA-1. On the other hand,SparkDrop-induced TIA-1 condensates do not recruit G3BP1 , and rapamycin alone(lack of FKBP or Frb) did not induce TIA-1 phase separation . Taken together, our dataindicates that the SparkDrop-induced phase separation of the SG scaffoldprotein G3BP1 can recruit the SG client proteins FUS and TIA-1 butnot vice versa. Recently, many SG proteins that interact with G3BP1have been identified,41 and the SPARK-ON-basedtools will be useful to further characterize these proteins for amechanistic understanding of SG formation and composition.We also conductedsimilar experiments for another SG client proteinTIA-1, which showed similar results that SparkDrop-induced G3BP1 condensatesrecruit TIA-1  of ∼5 min . As a control, we showed that DMSO did not induceYAP condensate formation. Furthermore, lenalidomide alone could notinduce YAP phase separation, using the YAP/SPARK-ON control (withoutHOTag6) . As a control,lenalidomide itself did not induce the condensate formation of YAP.These data indicate that the YAP condensates compartmentalize DNA-bindingpartner TEAD4 and have the potential to activate gene transcription.Next, we determined that the SPARK-ON-induced YAPcondensates interactand colocalize with the TEAD transcriptional factors using red fluorescentprotein mKO3-labeled TEAD4. Fluorescence imaging showed that, beforethe addition of lenalidomide, YAP was barely detected in the nucleus,whereas TEAD4 was in the nucleus but no TEAD4 was condensed 4B. After16 so that some MED1 puncta did not contain YAP/SPARK-ON.We first determined that the SPARK-ON-induced YAP condensates recruitedtranscriptional machinery. Because the mediator complex is requiredfor gene transcription by RNA polymerase II (RNAPII), we first examinedwhether the SPARK-ON-induced YAP condensates recruit MED1 (mediatorof RNA polymerase II transcription subunit 1). Immunofluorescenceimaging showed that these YAP condensates did colocalize with MED1condensates 4C. Some Supporting Figure S13), sitesof abundant rRNA transcription, reported to be stained intensely with5-EU.49 As a control, the HOTag3/SPARK-ONdroplets without YAP did not contain nascent RNA .Next,we determined that the SPARK-ON-induced YAP condensates containednascent RNA. Here we labeled nascent RNA by incubating the cells withuridine analog 5-ethynyluridine (EU) for 1 h so that EU is incorporatedinto newly transcribed RNA. The EU-labeled nascent RNA is detectedby using a copper (I)-catalyzed cycloaddition reaction  with azides labeled with red fluorescent dyes. Fluorescenceimaging in the red channel revealed several punctate structures 4D. The sCTGF using reverse transcriptionquantitative real-time PCR (RT-qPCR). Here we expressed YAP/SPARK-ON,incubated the cells with lenalidomide that activated SPARK-ON, andinduced YAP condensate formation. As a control, we incubated cellswith DMSO. Our data showed that the YAP condensates upregulated CTGF mRNA by ∼2.8-fold relative to that of the DMSOcontrol . Furthermore, thelenalidomide-activatable SPARK-ON was able to control YAP activityin a quantitative manner in regulating the expression of the targetgene CTGF by applying different levels of lenalidomide. As a positivecontrol, we incubated cells with sorbitol, which is known to induceosmotic stress and drive YAP condensate formation. This upregulatedthe CTGF mRNA level relative to that of the untreatedcells. Taken together, our results demonstrate that the lenalidomide-activatableSPARK-ON can manipulate YAP phase separation and the induced condensatesare transcriptionally active, containing DNA-binding partner TEAD,transcriptional machinery MED1, and nascent RNAs and upregulatingYAP target gene transcription.Finally, we determined that the SPARK-ON-inducedYAP condensatesare transcriptionally active and upregulated mRNA levels of a coreYAP target gene control 4F.We ve18 here we used SPARK-ONto examine role of YAP phase separation in gene transcription. Weengineered nuclear localized YAP (nlsYAP) with SPARK-ON. This nlsYAP/SPARK-ONwould maintain protein level of YAP in the nucleus, which can enableus to decouple role of PS from change of protein level in the nucleus.While a previous study concluded that YAP phase separation playeda role in gene transcription, it used a problematic approach by deletingthe activation domain,First, we demonstrated that lenalidomide activated SPARK-ON andinduced condensate formation within 3 to 4 min 5A. And tCTGF using DNA FISH (Supporting Excel File 1). These nlsYAP condensates also contained transcriptional machineryincluding MED1 and Pol II S5p so that upon phase separation the protein abundance level of TAZ inthe nucleus would not change. This will enable us to decouple therole of phase separation from the change in protein abundance in thenucleus. First, we demonstrated that SPARK-ON was able to drive thephase separation and condensate formation of TAZ in the nucleus. Lenalidomideactivated SPARK-ON that drove TAZ phase separation, forming condensatesin the nucleus within 2 min 6A,B. As Next, we determined that the nlsTAZ/SPARK-ON condensatescontainedthe DNA-binding partner TEAD. Here, live-cell fluorescence imagingrevealed that the SPARK-ON-induced TAZ condensates colocalized withthe condensed TEAD4 structures 6D. BeforCTGF. We labeledthe genomic DNA of CTGF using DNA FISH (fluorescencein situ hybridization).50 Confocal fluorescenceimaging revealed that the TAZ condensates were associated with thegenomic DNA of CTGF (Supporting Excel File 1). These data suggest that the TAZ condensates bind genomicDNA, consistent with the above results that these condensates containthe DNA-binding partner TEAD  based on immunofluorescence imaging 6F. Some Second, these condensates also contained RNA polymerase II (PolII) S5p 6G.FinalTaken together, these dataindicate that the SPARK-ON-induced TAZcondensates are transcriptionally active, containing the DNA-bindingprotein TEAD, genomic DNA, transcriptional machiner, and nascent RNA.CTGF and CYR61, two core target genes of TAZ, were significantly higher for thecondensed TAZ than for the dilute TAZ . As a control, we also prepared cells expressing the nlsTAZ/SPARK-ONcontrol (no HOTag6), which did not form condensates upon additionof lenalidomide. And lenalidomide alone did not increase the transcriptionof the two target genes.The above demonstration that the SPARK-ON-inducedTAZ condensates are transcriptionally active paved the way for usto investigate the role of phase separation. We first prepared cellsexpressing nlsTAZ/SPARK-ON and treated them with or without lenalidomide,which showed the condensed or dilute phase of TAZ, respectively 6I. And Wlute TAZ 6K. TheseTherefore, our results suggest thatTAZ phase separation, which can be regarded as a spatial reorganizationof the transcription factor, enhances the transcription of its targetgenes. Our work demonstrates that this lenalidomide-activatable SPARK-ONenables us to decouple the role of phase separation in gene transcriptionfrom the change in the protein level, which will be well suited forinterrogating the role of phase separation for many transcriptionfactors.52 The earlygroundbreaking work establishes that PS drives the formation of liquid-likecondensates2 and that multivalent interactionsdrive PS.3 Currently, there are two basicbiological questions regarding biomolecular condensates. First, dothey have biological activities? Increasing evidence suggests thatmany biocondensates are biologically active in cell signaling.9 For example, the YAP and TAZ condensates are transcriptionally activeand contain transcriptional machinery.20The condensate biology has been an excitingarea in cell and molecularbiology over the past decade because it sheds light on a new structuralentity that may play important roles in various cellular processes.21 Many biomolecules, including transcription factors,form condensates via PS when protein levels reach above the saturationconcentration. Biomolecular condensate formation can thus be dividedinto two steps: (1) the protein level increase above the saturationconcentration and (2) phase separation via weak and multivalent interactionsthrough IDRs. During phase separation, the biomolecules change froma diffuse state to a phase-separated state. The role of phase separationshould thus be determined by comparing biological activities betweenthese two states. An increase in the protein level such as a transcriptionfactor is known to affect transcription. Therefore, to understandthe role of phase separation, it is critical to decouple phase separationfrom the change in protein levels.Thesecond key question for the condensate biology field is whetherphase separation confers new or additional activities or is it simplya consequence of the protein level increase or molecular changes suchas post-translational modifications? This question remains mostlyunanswered due to conceptual and technical challenges.18 However, this approach is often problematic because the IDRs thatmediate PS are often within the activation domains that are essentialto transcription. For example, the activation domain of GCN4 is knownto interact with the mediator, and thus the mutations that are introducedinto the AD of GCN4 may possibly affect the interaction with the transcriptionalmachinery.58 Another example is the workon YAP phase separation. One conclusion from this work is that thephase separation of YAP played key roles in the transcriptional regulationof YAP target genes.18 Unfortunately, theapproach used in that work is problematic because in order to blockPS the AD was deleted, while blocked PS would also largely reducethe transcriptional activity of YAP because AD is essential to genetranscription and thus its deletion would also affect the diffusestate of YAP.Previously, mutagenesis-basedapproaches have been used to blockPS in order to determine the role of phase separation.de novo-designed coiled coils, we engineered the SPARK-ON tool that is appropriatefor manipulating protein phase separation because first, the operatingphysical principle of SPARK-ON is based on multivalent PPIs that havebeen shown to drive PS,3 second, the SPARK-ON-inducedcondensates are highly dynamic and possess liquid-like properties,and third, the SPARK-ON tag exerts no or little perturbation on thefunction of transcription factors including YAP and TAZ. For example,our data show that SPARK-ON-tagged YAP and TAZ are both transcriptionallyactive and regulate the transcription of their core target gene CTGF. Furthermore, SPARK-ON-based manipulation of TAZ phaseseparation reveals the role of PS in promoting the transcription of CTGF, which is consistent with a previous study.20 On the other hand, our work clearly showed thatPS of YAP promotes the transcription of the YAP target genes, reachinga similar conclusion with the previous work,18 though the previous work was based on the problematic approach asdiscussed above. Therefore, using SPARK-ON chemogenetic tools, wehave obtained important biological findings on the role of phase separationof transcription factors on regulating gene expression.To address the above challenge and decouple PSfrom protein-levelchanges without introducing mutations, we have developed chemogenetictool SPARK-ON, which decouples phase separation from protein abundance.We first applied the structure-based approach and engineered a newchemogenetic heterodimer that is inducible by FDA-approved drugs collectivelyknown as IMiDs. This new tool is complementary to and has severaladvantages over the FKBP-Frb pair-based systems in manipulating PS,including a smaller size  and a weaker interaction thatbetter mimics weak interaction in most biomolecular condensates. Byincorporating the multivalent tags from the 53 OurSPARK-ON-based approach further reveals that phase separation of theclient proteins including FUS and TIA1 does not recruit G3BP1. Therefore,our results, together with others, support the scaffold–clientmodel of condensate assembly in which the phase separation of a scaffoldrecruits clients but not vice versa.34 Additionally,it has been reported that the optogenetic tools are not successfulin manipulating full-length FUS and TIA1, though they were able todrive the condensate formation of truncated mutants containing LCDor IDR.54 The manipulation of full-length FUS andTIA1 phase separation is preferred since the LCD is not the entireprotein after all.55 One advantage of usingthe chemogenetic systems is that it allows us to combine two chemogeneticsystems so that we do not need to fuse the multivalent tag (HOTag)to proteins of interest , which maintains the stoichiometryof the protein before the induced phase separation.We alsodemonstrated that SPARK-ON can be used to manipulate proteinsother than TFs. We applied SPARK-ON to manipulate stress granule proteinsincluding G3BP1, FUS, and TIA1. We combined our lenalidomide-induciblechemogenetic tool with the rapamycin-inducible system in manipulatingprotein phase separation because the two orthogonal small molecule-inducibleheterodimers are compatible and can be combined to control proteininteraction and phase separation. In particular, we used lenalidomide-inducibleSPARK-ON and the rapamycin-inducible PPIs to interrogate the scaffold–clientmodel of SG assembly. Our data indicate that the phase separationof SG scaffold protein G3BP1 recruits its clients including FUS andTIA1, which is consistent with the previous results using optogenetics-basedapproaches in manipulating G3BP1.In summary,the engineered chemogenetic SPARK-ON complements andoffers several advantages to other approaches, including optogeneticsand mutagenesis, in dissecting the role of phase separation in biologicalfunctions, which is one of the key basic questions in condensate biology.SPARK-ON can be applied to cytosolic proteins and transcription factors.Our work supports the scaffold–client model of SG assemblyand reveals that the phase separation of YAP/TAZ promotes gene transcription.Therefore, the SPARK-ON chemogenetic tool is a versatile tool forthe condensate biology field in dissecting the condensate assemblymechanisms and functional roles in cells.


Here, we provide support for the alternative nunatak refuge hypothesis—that ice-free terrain with sufficient relief above the ice sheet provided refuges and was a source for terrestrial biota found today. This hypothesis is supported here by an increased understanding from the combination of biological and geological evidence, and we outline a mechanism for these refuges during successive glacial maxima that also provides a source for coastal species. Our cross-disciplinary approach provides future directions to further test this hypothesis that will lead to new insights into the evolution of Antarctic landscapes and how they have shaped the biota through a changing climate.The origin of terrestrial biota in Antarctica has been debated since the discovery of springtails on the first historic voyages to the southern continent more than 120 years ago. A plausible explanation for the long-term persistence of life requiring ice-free land on continental Antarctica has, however, remained elusive. The default  The first springtails collected were during the Belgian Antarctic Expedition 1897–1899) from Harry Island (Antarctic Peninsula) . Springt–1899 frostracods –19. Two stracods ,5), and te (e.g. ).nunatak refuge hypothesis—that the survival of the vast majority of biota during glacial maxima occurred on terrain that protruded through the ice sheet (nunataks) with sufficient relief to have remained ice-free—has gained support based only on the unique assemblage of biota present [geothermal activity was proposed as a solution [nunatak refuge hypothesis. Importantly, we have compiled support by using an increased understanding from the combination of biological and geological evidence, and we detail a mechanism that provides an essential source for coastal species.More recently, the alternative  present ,10,22. Dsolution . Here, w. 2 (a)coastal regions of Antarctica [et al. [geothermal activity provides a mechanism to maintain ice-free land near coastal margins during glacial maxima. For geothermal activity to provide a plausible mechanism maintaining ice-free coastal refuges, it would require there to be sufficient sites throughout Antarctica where regionally isolated endemic species would have persisted across the LGM and into the present. Fraser et al. [do not overlap with any geothermal site  (i)nunatak refuge hypothesis to be robust, we need to explore if variations in ice sheet thickness across Antarctica during glacial maxima allowed for widespread ice-free habitats to be maintained. Modern ice sheet reconstructions based on both onshore and offshore geological evidence, as well as dating of these features shows that the ice sheet at the LGM expanded onto the continental shelf and in coastal regions it thickened by hundreds of metres [figure 2a; electronic supplementary material, figures S1–S7). Most of these regions harbour short-range endemic biota, including springtails (figure 2a). In some locations, such as Victoria Land, Transantarctic Mountains and Dronning Maud Land, there is evidence of ice-free conditions persisting for millions of years [figure 2a; electronic supplementary material, figures S3, S6 and S7). Such marine-based glaciers are known to be highly dynamic, substantially expanding and contracting during glacial-interglacial cycles  prov (ii)a; see also electronic supplementary material, file). So how does this explain species presence today in coastal habitats? Clues to this remarkable survival come from well-known alpine and polar studies  in terrain that protruded above or on the modern ice sheet (figure 2b). During an interglacial, species disperse down slopes and valleys with the glacial foreland that shifts with glacial margins as the ice retreats (figure 2c), with dispersal also potentially being assisted by meltwater . The converse is also true; springtails are absent from extensive ice-free regions (see figures 2a), likely because these regions do not contain terrain with appropriate environments to serve as refuges when lowland ice expanded during glacial maxima.For Antarctic biota, there is a growing body of molecular evidence revealing long-term isolation and persistence of short-range endemic species on the Antarctic continent ,58–64. Ffigure 2es e.g. –68). In . In a; ses  spreadi. 3test predictions using an evidence-based approach. We posit that Antarctica's nunataks provide the most likely refuge for the long-term survival of species, and this explains the patterns of isolated short-range endemic species we find today, where there is an increasing body of evidence that none are shared between regions and some regions are entirely lacking in species. The nunatak refuge hypothesis also explains how species can be present during interglacials in coastal areas adjacent to their refugia—they do so by shifting up and down slopes within glacial foreland ecosystems.Our suggested framework provides the necessary way forward to cross-disciplinary collaborations can provide a more complete picture of the evolution of Antarctic landscapes and the biota they harbour, but this requires cosmogenic isotope dating specifically targeted towards identifying ice-free terrain at the LGM or older (e.g. [a), there are three specific tests that can be carried out to further evaluate the nunatak refuge hypothesis: (1) the Antarctic Peninsula has well-documented springtail records, but their provenance is uncertain [a).Going forward, er e.g. ,43,45), ,45, croser  and geoncertain ,77), butncertain —such bioncertain ,36,47–51. 4We have described a mechanism that allowed for springtails to survive in isolation in refugia across the LGM in ice-free habitats. Support for long-term continuity across Antarctic ice-free terrain is provided by geological dating of glacial deposits since the ice sheet first formed . MorRecent modelling of snow and ice melt indicates that current ice-free regions in Antarctica may expand up to 25% within the twenty-first century . If thes


Ankrd31 gene that reduce or eliminate the interaction with REC114 without altering interactions with the other known ANKRD31 partners. Analysis of these mice demonstrates that the ANKRD31–REC114 interaction is critical for all ANKRD31 functions in meiosis.Recombination between homologous chromosomes is essential for gamete formation in mammals. Recombination initiates with DNA double-strand breaks (DSBs), whose formation is tightly regulated. The vertebrate-specific ANKRD31 protein is an important part of this regulation in mice and humans and is particularly important for ensuring that the sex chromosomes in males can recombine. ANKRD31 interacts with many different proteins including the REC114 protein, but it has not been established whether these interactions are important for meiosis. We generated mice with targeted mutations in the  Ankrd31 mutations that either reduced (missense mutation) or eliminated  the ANKRD31–REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31–REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31–REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114 and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that “anchors” other proteins together and to meiotic chromosomes. To determine whether and why the REC114 interaction is important for ANKRD31 function, we generated mice with  During meiosis, one round of DNA replication is followed by two rounds of chromosome segregation to reduce the chromosome complement. In many species, the segregation of homologous chromosomes during meiosis I requires programmed DNA double-strand breaks (DSBs) to be generated and then repaired by homologous recombination. These DSBs are formed by SPO11 and accessory proteins –5. AlthoSI Appendix, Fig. S1A). Other segments in ANKRD31 interact with additional partners IHO1, MEI1, PTIP, and ZMYM3 (The vertebrate-specific protein ANKRD31 (Ankyrin Repeat Domain Containing 31) was identified as a direct interaction partner of REC114 , 7. ANKRnd ZMYM3 –8  of the X and Y chromosomes, which are necessary for sex chromosome pairing, recombination, and segregation in spermatocytes  to generate mutations that disrupt the ANKRD31–REC114 interaction to different extents. Analysis of mice carrying Ankrd31 mutant alleles in various combinations showed that this interaction is essential for all ANKRD31 functions, with specific functions dependent to different quantitative degrees on the strength of the interaction.To address these issues, we used CRISPR/Cas9 genome editing to alter the last C) complexed with REC114 residues 1–158 (REC114N), the side chain of ANKRD31 Glu-1831 is anchored by two intermolecular salt bridges and one hydrogen bond . In pulldown assays with recombinant GST-tagged ANKRD31C and purified REC114N, substituting alanine for Glu-1831 dramatically reduced the interaction .In the crystal structure for ANKRD31 residues 1808–1857 (ANKRD31gen bond  (SI Appeeraction . This muEAAnkrd31). We also fortuitously obtained a truncated allele (Ankrd31ΔC) with a 1-bp deletion at position 5,484 that results in a frameshift mutation and premature termination, replacing the last 21 amino acids with 9 extraneous residues  suggests that there may be additional contacts between REC114N and other parts of ANKRD31  but significantly heavier than Ankrd31ΔC/ΔC and –/–Ankrd31 males , and males of both genotypes were able to sire pups, although we cannot rule out that they were subfertile.To explore whether there is a dosage effect of the SI Appendix, Fig. S2B). In contrast, –/–Ankrd31 tubules, as was previously shown . In contrast, the majority of EA/EAAnkrd31 tubules appeared normal, and only a small fraction appeared abnormal (emptier and lacking postmeiotic cells). Compound heterozygotes EA/–Ankrd31 and Ankrd31EA/ΔC both had some normal tubules but also had a larger fraction of abnormal tubules as compared to EA/EAAnkrd31 .Testis sections from control animals had the full array of spermatogenic cells, including spermatocytes and round and elongated spermatids, as expected  of apoptotic tubules, and the compound heterozygotes again showed an intermediate phenotype (~20% apoptotic tubules) . In all mutants tested, dying spermatocytes were observed in both pachynema and metaphase I, indicating varying penetrance of arrest at both stages of prophase I  of apoptotic tubules in I arrest , 7 . Seeing fewer RPA2 foci in early cells suggests that the reduction of RAD51 and DMC1 foci is caused by a delay and/or reduced efficiency of forming cytologically observable sites containing resected DSBs, not simply because of defective loading of the strand-exchange proteins. Thus, it has been interpreted that there is a delay in global DSB formation in the absence of ANKRD31 ; these are interpreted as signs of persistent DSBs, suggesting a defect in DSB repair . We conclude that the ANKRD31–REC114 interaction is essential for ANKRD31 functions in global DSB formation and recombination.EA/EAAnkrd31 mice showed a similar but quantitatively milder defect. Compared to control mice, EA/EAAnkrd31 spermatocytes had reduced γH2AX staining throughout early prophase I . Again, EA/–Ankrd31 andAnkrd31EA/ΔC compound heterozygotes showed an intermediate phenotype, with reduced γH2AX staining throughout early prophase I  and even fewer DMC1, RAD51, and RPA foci than the EA/EAAnkrd31 homozygous mice .ophase I D and E. SI Appendix, Fig. S4B). We conclude that attenuating the ANKRD31–REC114 interaction causes a delay in global DSB formation and a defect in completion of recombination that are both quantitatively more modest than when the interaction is more completely disrupted.In all cases, focus numbers declined as chromosomes synapsed, but to different extents, resulting in more or less similar numbers of foci at late zygonema for all of the genotypes that include the missense allele. However, pachytene cells with apparently normal synapsis also had elevated numbers of DMC1 and RAD51 foci, with the degree of elevation correlating with the severity of the REC114 interaction defect . This analysis did not reproduce the small but statistically significant increase .We additionally examined crossing-over on autosomes by staining for MLH1, a component of the Holliday junction resolvase , 29. Pacincrease  or decreincrease  in MLH1 –/–Ankrd31 metaphase I cells frequently had unaligned chromosomes, and sex chromosomes failed to pair in 92% of otherwise normal-looking pachytene cells  and 89% (8 out of 9) pachytene cells with paired XY were in early/mid pachynema, respectively. In comparison, in wild type or heterozygous controls, 54% (32 out of 59) and 46% (27 out of 59) of pachytene cells with paired X and Y were in early/pachynema and late pachynema, respectively.In keeping with the similarly high frequency of metaphase I arrest as krd31–/– G, Ankrd3achynema . These dEA/EAAnkrd31 had a much more modest defect, with only 20.6% of sex chromosomes unpaired, while EA/–Ankrd31 and Ankrd31EA/ΔC compound heterozygotes again had an intermediate defect, with about 50% of sex chromosomes unpaired . We therefore asked whether the ANKRD31–REC114 interaction itself is specifically required. When chromosome spreads were stained for REC114, blobs were reduced in number and intensity in all of the n defect A–C. ANKRn defect D–F.Ankrd31 but are reduced in number and intensity during leptonema and early zygonema  such as transcription promoters and CpG islands . Two biological replicates were generated for each genotype and averaged .To do this, we performed Exo7/T-seq  in cy (POI) . Both vaANKRD31  carries insertion of an A residue in exon 3, causing frameshift and premature termination (SI Appendix). Mice were genotyped using PCR on cut toes lysed in Direct Tail lysis buffer (Viagen) with primers as indicated in SI Appendix, Table S2.Mouse experiments were approved by the Memorial Sloan Kettering Cancer Center (MSK) Institutional Animal Care and Use Committee (IACUC) and followed US federal regulatory standards. The previously described Ankrd31ΔC/ΔC mice, we used testes of younger mutant mice (1.5 mo old) to assess protein levels (Left). We used adult testes for ANKRD31-EA (Right) because the missense mutation causes less of a reduction in testis weight. The slight decrease in ANKRD31-EA levels compared to the littermate control may be a consequence of the smaller testes in the mutant. Given that the mutants have much smaller testes and fewer spermatocytes (more apoptotic spermatocytes) as compared to wild type, our observation of comparable or only slightly reduced protein expression supported our conclusion that the mutations did not grossly destabilize ANKRD31 protein. Testis extracts were treated with Benzonase to degrade DNA and immunoprecipitated with  and ProSI Appendix, Methods). Slides were blocked for 30 min at room temperature in a Coplin jar with 100 mL fresh block solution [1× PBS with 0.05% Tween-20 and 3 mg/mL bovine serum albumin (BSA)]. Slides were incubated with 80 µL primary antibody in block solution and covered by parafilm overnight at 4 °C in a humid chamber. Slides were washed 3 × 5 min in block solution, then incubated with 80 µL secondary antibody in block solution for 30 min in a humid chamber at room temperature. Slides were then washed 3 × 5 min in the dark with block solution, rinsed in milliQ H2O, and mounted with Vectashield containing DAPI. Primary and secondary antibodies are listed in SI Appendix, Table S3. Images were acquired on a Zeiss Axio Observer Z1 Marianas Workstation, equipped with an ORCA-Flash 4.0 camera, illuminated by an X-Cite 120 PC-Q light source, with 100 × 1.4 NA oil immersion objective. Marianas Slidebook  software was used for acquisition.Chromosome spreads were prepared by hypotonic lysis in the presence of paraformaldehyde from cell suspensions generated by enzymatic digestion of decapsulated testes with collagenase and trypsin  blur was used to isolate REC114 or ANKRD31 foci. Scripts are available on Github: For γH2AX intensity analysis, cells were substaged manually by SYCP3 staining. Regions of interest were drawn, and integrated density was measured. A region containing no cells was measured for background subtraction. To combine measurements from different experiments, the integrated density was normalized to the mean integrated intensity of wild-type leptotene cells within each experiment.E. coli exonuclease VII and exonuclease T to remove ssDNA, like END-seq, but uses the methods for genomic DNA preparation, adapter ligation, and sequencing library preparation from S1-seq. Full details are provided in SI Appendix. Briefly, high-molecular-weight genomic DNA was extracted from enzymatically dissociated testicular cells embedded in agarose plugs and then digested with exonuclease VII and exonuclease T. Biotinylated adapters were ligated to the blunted ends; then, the DNA was extracted from the plugs and sheared by sonication, and ligated fragments were purified using streptavidin beads. A second adapter was ligated to the sheared end of each fragment, and the resulting DNA library was amplified by low-cycle-number PCR and then sequenced on the Illumina HiSeq platform in the Integrated Genomics Operation at MSK. We obtained paired-end reads of 50 bp.Exo7/T-seq combines elements of previously described S1-seq , 32 and SI Appendix, Fig. S6D). Therefore, to compare spatial patterns of resection in SI Appendix.Bioinformatic analysis was performed as described previously , 32. BriAppendix 01 (PDF)Click here for additional data file.Dataset S01 (XLSX)Click here for additional data file.


Sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown promise in reducing the risk of atrial fibrillation (AF). However, the results are controversial and the underlying metabolic mechanism remains unclear. Emerging evidence implied that SGLT2 inhibitors have extra beneficial metabolic effects on circulating metabolites beyond glucose control, which might play a role in reducing the risk of AF. Hence, our study aimed to investigate the effect of circulating metabolites mediating SGLT2 inhibition in AF by Mendelian randomization (MR).SLC5A2 gene and glycated hemoglobin level (HbA1c). Positive control analysis on type 2 diabetes mellitus (T2DM) was conducted to validate the selection of genetic instruments.A two-sample and two-step MR study was conducted to evaluate the association of SGLT2 inhibition with AF and the mediation effects of circulating metabolites linking SGLT2 inhibition with AF. Genetic instruments for SGLT2 inhibition were identified as genetic variants, which were both associated with the expression of P = 0.006) and AF . Among 168 circulating metabolites, two metabolites were both associated with SGLT2 inhibition and AF. The effect of SGLT2 inhibition on AF through the total concentration of lipoprotein particles  and the concentration of HDL particles , with a mediated proportion of 8.03%  and 7.59%  of the total effect, respectively.Genetically predicted SGLT2 inhibition (per 1 SD decrement in HbA1c) was associated with reduced risk of T2DM (odds ratio [OR] = 0.63 , This study supported the association of SGLT2 inhibition with a reduced risk of AF. The total concentration of lipoprotein particles and particularly the concentration of HDL particles might mediate this association. Further mechanistic and clinical studies research are needed to understand the mediation effects of circulating metabolites especially blood lipids in the association between SGLT2 inhibition and AF.The online version contains supplementary material available at 10.1186/s12933-023-02019-8. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a class of oral antidiabetic drugs, which reduce serum glucose concentration by inhibiting the reabsorption of glucose in proximal tubules and promoting urinary glucose excretion.  CompelliSGLT2 inhibitors were believed to have extra beneficial metabolic effects beyond glycemic control, , 11 whicMendelian randomization (MR) is a powerful approach that uses genetic variants associated with exposure as instruments to examine the potential causal association between exposure and outcome. MR is less likely to be affected by confounding or reverse causality,  as it miIncreasing evidence suggested a correlation between metabolism disorders and the occurrence of AF. , 22 In aTherefore, we hypothesized that circulating metabolites might mediate the effect of SGLT2 inhibition on AF. In the present study, we first conducted a two-sample MR to examine the association between SGLT2 inhibition and AF. Second, we performed a two-step MR study to establish the potential metabolic pathway from SGLT2 inhibition to AF through circulating metabolites, particularly blood lipids. We investigated the possible causal effect of SGLT2 inhibition on circulating metabolites, which would provide insights into the metabolic mechanism linking the effect of SGLT2 inhibition with AF.The current study was conducted using a two-sample MR design Fig. . To ensuSLC5A2 (gene of SGLT2). Second, we estimated the associations of each SLC5A2 variant with glycated hemoglobin (HbA1c) level, an indicator of glucose-lowering effect, and selected variants that are significantly associated with HbA1c (P < 1 × 10− 4). The genome-wide association study (GWAS) data of HbA1c was from 344,182 unrelated individuals of European ancestry without diabetes mellitus in the UK Biobank  and were independent of each other .We systematically searched 249 nuclear magnetic resonance circulating metabolites from 121,000 European ancestry participants generated by Nightingale Health.  These meWe obtained publicly available GWAS summary data for AF from Atrial Fibrillation Genetics (AFGen) Consortium  (Supplementary Table We applied two-sample univariable MR (UVMR) to estimate the effect of SGLT2 inhibition on AF and T2DM. Before MR analysis, we used Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO)  and radiTwo-step MR was conducted to estimate the mediation effect of circulating metabolites on the association between SGLT2 inhibition and AF Fig. b and c. In UVMR analysis assessing the effects of SGLT2 inhibition on AF and T2DM, we performed the MR-Egger, MR-PRESSO, weighted median, simple mode, and weighted mode methods as sensitivity analyses. The MR-Egger method examines whether there is directional pleiotropy based on its intercept term, where a value that differs from zero indicates that the presence of directional pleiotropy and the IVW estimate is biased.  In additThe strength of the genetic instruments was assessed by F statistics and indicates weak instruments when F statistics < 10. Cochrane’s Q statistics for IVW and the global test for MR-PRESSO were calculated to evaluate the heterogeneity between the instruments.The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.P value that passed the Bonferroni corrected P value was defined as statistically significant, but P value < 0.05 was considered statistically significant for the effect of SGLT2 inhibition on T2DM and AF since the outcome T2DM was not the primary outcome in the present study.All MR analyses were conducted with the package “TwoSampleMR”, “MendelianRandomization”, “MVMR”, “MRPRESSO”, and “RadialMR” in R software (version 4.2.2). To control for multiple testing, a two-sided P = 0.006) and AF , for per 1 SD lowering of HbA1c via SGLT2 inhibition , and no horizontal pleiotropy was detected .In total, 10 independent single nucleotide polymorphisms (SNPs) were selected as genetic instruments for SGLT2 inhibition and the F statistics for all SNPs were greater than 16  (Supplementary Table P = 2.23 × 10−5). For lipoprotein particles, we found that SGLT2 inhibition increased the concentration of HDL particles especially small HDL  but decreased the concentration of very large HDL particles .We estimated the effect of SGLT2 inhibition on the 168 circulating metabolites and observed 20 metabolites were significantly associated with SGLT2 inhibition  (Supplementary Table P = 0.002). For the concentration of HDL particles, we also observed a negative association with AF . These results were supported by the MR-PRESSO method. There was no evidence of heterogeneity  and no horizontal pleiotropy . The genetic variants for the 20 metabolites were all strong (F statistics > 23) (Supplementary Data 1–20 are available at 10.6084/m9.figshare.23731140).We further estimated the effect of the 20 circulating metabolites that were significantly associated with SGLT2 inhibition on AF and found that two metabolites were significantly associated with AF (Bonferroni-corrected P = 0.004) , with a mediated proportion of 8.03%  of the total effect (Table P = 0.005) had a mediated proportion of 7.59% . There was no evidence of heterogeneity and no horizontal pleiotropy in these associations.We observed an indirect effect of SGLT2 inhibition on AF through the total concentration of lipoprotein particles  and AF . The total concentration of lipoprotein particles might mediate 8% of the effect of SGLT2 inhibition on AF, particularly with the concentration of HDL particles mediating 7.6%.In the present study, we evaluated the associations between genetically predicted SGLT2 inhibition and T2DM and AF. Furthermore, we investigated the mediating role of circulating metabolites in the association between SGLT2 inhibition and AF. Our study indicated that genetic variation in SGLT2 inhibition targets was associated with a lower risk of T2DM  in patients with T2DM. [8, 0.95) k of AF. , 54.The effects of SGLT2 inhibitors on blood lipids are conflicting in previous studies. , 56 A reIn our study, we provided insights into the effect of SGLT2 inhibitors on lipid metabolism by using a set of robust genetic variants of SGLT2 as the instruments and the largest circulating metabolites GWAS to date, which mainly covered blood lipids and lipoprotein particles sub-fractions.  We obserOur findings support that genetically predicted total concentration of lipoprotein particles and concentration of HDL particles were associated with AF, which were consistent with previous findings from one meta-analysis  and seveTo our knowledge, this is the first study to use MR analysis to investigate the relationship between SGLT2 inhibition, circulating metabolites, and AF in the general population. Furthermore, we provided genetic evidence on the potential mechanism of SGLT2 inhibition exerting a beneficial effect on AF through the total concentration of lipoprotein particles, primarily HDL. Nevertheless, our study had several limitations. First, genetic variants mimicking SGLT2 inhibition reflect lifelong effects of SGLT2 inhibitors, and the magnitude of these effects might not accurately reflect the magnitude of short-term effects of SGLT2 inhibitors.  Hence, MIn conclusion, this study supported the association between genetically predicted SGLT2 inhibition, circulating metabolites, and AF. Specifically, the total concentration of lipoprotein particles, especially HDL particle concentration, mediated the protective effect of SGLT2 inhibition on AF. These findings provided genetic evidence for the mechanisms of SGLT2 inhibition in reducing AF risk and might inform future mechanistic and clinical studies.Below is the link to the electronic supplementary material.Supplementary Material 1


Irritable bowel syndrome with predominant diarrhea (IBS-D) and functional diarrhea (FD) are disorders of gut–brain interaction characterized by recurring symptoms which have a serious impact on the patient’s quality of life. Their pathophysiology is far from being completely understood. In IBS-D growing evidence suggests that bile acid malabsorption (BAM) could be present in up to 30% of patients. Microscopic colitis (MC) is a well-known cause of watery diarrhea and some patients, at first, can be diagnosed as IBS-D or FD. Both BAM and MC are often responsible for the lack of response to conventional treatments in patients labelled as “refractory”. Moreover, because BAM and MC are not mutually exclusive, and can be found in the same patient, they should always be considered in the diagnostic workout when a specific treatment for BAM or MC is unsatisfactory. In the present review the possible shared pathogenetic mechanisms between BAM and MC are discussed highlighting how MC can induce a secondary BAM. Moreover, a brief overview of the current literature regarding the prevalence of their association is provided. Irritable bowel syndrome with diarrhea (IBS-D) and functional diarrhea (FD) are the two major disorders of gut–brain interaction characterized by watery diarrhea , with a Lymphocytic Colitis (LC), in the presence of an increased number of intraepithelial lymphocytes  combined with an increased inflammatory infiltrate in the lamina propria and a not significantly thickened collagenous band (<10 mm).Collagenous Colitis (CC), in the presence of a thickened subepithelial collagenous band (≥10 mm) combined with an increased inflammatory infiltrate in the lamina propria.Some studies have estimated that up to one third of patients diagnosed as IBS-D or FD may actually have a bile acid diarrhea (BAD), and that about 1% of the general population suffers from bile acid malabsorption (BAM) ,9. FurthWhen the abovementioned histopathological diagnostic criteria are not fully matched, it is also possible to identify an incomplete MC .The overall prevalence of MC in the general population is 119/100,000 .In patients meeting the criteria for IBS-D or FD not responding to conventional therapy, all recent guidelines suggest that BAM should be ruled out through a positive diagnosis with a SeHCAT (75-selenium homocholic acid taurine) test . When Se75Se) after seven days; a retention value of ≤10–15% is usually considered diagnostic of BAM . AS. AS46]. ASBT is downregulated by FXR, reducing bile acid reuptake, with the possibility of developing BAD. Studies on mice have demonstrated that in the case of experimental colitis, induced with dextran sulfate sodium, inflammation increases the level of c-fos proteins, which causes a direct inhibition of ASBT. This leads to an increase in bile acids that arrive in the colon . MoreoveModulating intestinal inflammation. In a mice mode, the activation of TGR5 leads to an anti-inflammatory effect through a reduction in IL6, TNFα, INFγ and an increase in IL10.Maintaining intestinal barrier integrity. In a mice model, a deficit in TGR5 leads to an abnormal morphology of the colonic mucosa and increased intestinal permeability, with an altered molecular architecture of epithelial thigh junctions, increased expression and abnormal distribution of zonulin 1.Intestinal motility. It is expressed on enteric neurons and mediates the effects of bile acid on colonic motility. Confirming this hypothesis, a study by Alemi et al. showed that TGR5 overexpression in transgenic mice caused a more rapid colonic transit time and increased frequency of defecation .Another physiopathological aspect under debate is the TGR5 pathway. This is a membrane G protein-coupled bile acid receptor mainly expressed in the gastrointestinal tract. Its expression is higher in the distal ileum and colon, but it can be found also in cholangiocytes, stellate cells, sinusoidal endothelial cells and Kupffer cells of the liver. TGR5 is activated by bile acids and plays a role in insulin sensitivity, adipose tissue browning, reduction in hepatic steatosis. It is also involved in gallbladder filling and inflammation through inhibition of NFkB. TGR5 gene expression is partially regulated through FXR, indicating that FXR and TGR5 may interplay to regulate metabolism in the liver and gut . TGR5 seTherefore, a possible dysregulation of TGR5 induced by bile acids could play an etiopathogenetic role in inflammatory diseases of the colon , such asThe clinical management of IBS-D and FD may be complex and uncertain ,5,54. GuIt is important to perform multiple biopsies during colonoscopy while evaluating patients with chronic watery diarrhea, even when the colonic mucosa appears to be macroscopically normal, since this is the only way of reliably diagnosing MC.Exploring the exact prevalence of the association of BAM and MC in chronic watery diarrhea or in patients labelled as “functional” but refractory to conventional therapy;Clarifying the role of the most debated molecular actors  involved in BAM pathophysiology and its relationships with MC.Understanding the relationship between BAM and MC in symptom generation, when both coexist in the same patients;Discovering the correct treatment strategy in these patients through randomized controlled clinical trials.Further research should focus on:


Pubic ramus fractures account for the most common types of pelvic fractures. The standard surgical approach for superior pubic ramus fractures (SPRF) is a minimally invasive percutaneous screw fixation. However, percutaneous closed reduction and internal fixation of anterior pelvic ring injuries have high failure rates of up to 15%. The aim of this biomechanical study was to evaluate the stability of SPRF following stabilization with retrograde placed cannulated compression headless screw (CCHS) versus conventional fully and partially threaded screws in an artificial pelvic bone model.n = 6) for fixation with either a 7.3 mm partially threaded cannulated screw (group RST), a 7.3 mm fully threaded cannulated screw (group RSV), or a 7.5 mm partially threaded cannulated CCHS (group CCS). Each hemi-pelvic specimen was tested in an inverted upright standing position under progressively increasing cyclic axial loading. The peak load, starting at 200 N, was monotonically increased at a rate of 0.1 N/cycle until 10 mm actuator displacement.SPRF type II as described by Nakatani et al. was created by means of osteotomies in eighteen anatomical composite hemi-pelvises. Specimens were stratified into three groups of six specimens each (p ≤ 0.033). The differences between RST and RSV were significant for total displacement (p = 0.020), and a trend toward significance for torsional displacement (p = 0.061) was observed. For both failure criteria 2 mm total displacement and 5° torsional displacement, CCS was associated with significantly higher number of cycles compared to RST (p ≤ 0.040).Total and torsional displacement were associated with higher values for RST versus CCS and RSV, with significant differences between RST and CCS for both these parameters (CCHS fixation presented predominantly superior stability to the standard surgical treatment and could therefore be a possible alternative implant for retrograde SPRF screw fixation, whereas partially threaded screws in group RST were associated with inferior biomechanical stability. Fractures of the pubic ramus are among the most common pelvic fracture types and a demanding need for their improved management has been reported . There iMinimally invasive percutaneous screw fixation is one of the standard procedures for treatment of superior pubic ramus fractures (SPRF) recognized for shorter surgery times and reduced blood loss when compared to other fixation methods, such as plate fixation –11. HoweTherefore, the aim of this biomechanical study was to evaluate the stability of SPRF following stabilization with retrograde placed cannulated compression headless screw (CCHS) versus conventional fully and partially threaded screws in an artificial pelvic bone model. The hypothesis was that CCHS would provide equivalent or greater stability when compared to conventional ramus screws. To our knowledge, this surgical technique of CCHS fixation of SPRF has never been evaluated.Eighteen anatomical composite hemi-pelvises  were used in this study. A type II fracture was created in each specimen at the middle zone of the superior pubic ramus according to the Nakatani classification system  by meansn = 6) with equal distribution of left and right anatomical sites for instrumentation as follows:Group RST: SPRF stabilization using 7.3 mm partially threaded cannulated screw, length 90 mm , while those in group CCS were made from titanium alloy (Ti-6Al-4 V). An experienced surgeon with senior consultant status performed all procedures.Biomechanical testing was performed on a servohydraulic material test system  equipped with a 4 kN load cell . The setup used to mount the specimen for testing was adopted from previous studies investigating ramus fracture fixation  Fig. 3)3). Each Axial compression along the machine axis was applied to the acetabulum via a ceramic ball of 28 mm radius. Homogenous load transfer to the specimens was achieved by a molded PMMA hemispherical cavity inserted in the acetabulum. The setup configuration targeted a simulation of hip joint reaction force trajectory during walking, as described by Bergmann et al. .The loading protocol commenced with an initial nondestructive quasi-static ramp from 20 N preload to 200 N at a rate of 18 N/s, followed by progressively increasing cyclic loading in axial compression with a physiological profile of each cycle at a rate of 2 Hz . KeepingMachine data of axial displacement and axial load were continuously acquired from the machine transducer and load cell throughout the tests at 200 Hz. Based on these, the construct stiffness was calculated from the ascending load–displacement curve of the initial quasi-static ramp within the linear loading range between 80 and 180 N.The relative movements between the fractured fragments were continuously assessed at 20 Hz throughout the tests in all six degrees of freedom by means of optical motion tracking. For that purpose, an individual marker set, consisting of multiple single optical markers, was attached to the superior and inferior fragment adjacent to the fracture line using Kirschner (K-) wires. A local anatomical coordinate system was created based on proper alignment of the superior marker set. A second coordinate system was created based on the fracture plane, which was virtually defined using a dedicated touch probe. The coordinates of the markers were tracked with a stereographic optical camera system  within these two coordinate systems. Based on these measurements, the relative movements of the two most anterior aspects—each one associated to either fragment defined by the attached marker sets, but initially overlapping each other at the reduced fracture state—relative to each other, were calculated as the Euclidean normal distance of the translational displacements along the three principal axes and defined as total displacement. In addition, the combined angular displacement was calculated as the gap opening between the two initially reduced osteotomy/fracture surfaces adjoining each other in the fracture gap and defined as gap angle. Furthermore, the angular displacement between the fragments in the fracture plane was assessed and defined as torsional displacement.The outcome measures were calculated at five intermittent time points of cyclic testing after 2000, 4000, 6000, 8000, and 10,000 test cycles. The latter represented the highest rounded number of cycles at which none of the specimens had failed and dropouts could not artifactually influence the results. The values were considered with respect to the values at the beginning of the cyclic test and were calculated in peak loading condition. Additionally, three different criteria for specimen failure were set at 2 mm total displacement, 5° gap angle, as well as 5° torsional displacement, and the corresponding number of cycles until fulfillment of these criteria were calculated.Statistical analysis among the outcome measures was performed with SPSS software . Mean and standard deviations were calculated for each parameter of interest and group separately. General linear model repeated measures test with Bonferroni post hoc test for multiple comparisons was conducted to determine significant differences between the treatment groups for the outcome measures of the parameters of interest evaluated over the time points during cyclic testing after 2000, 4000, 6000, 8000, and 10,000 cycles. Initial axial construct stiffness and the numbers of cycles for the different failure criteria, including cycles to earliest failure, were compared among the groups with one-way analysis of variance (ANOVA) considering either Bonferroni or Games-Howell post hoc tests. Level of significance was set at 0.05 for all statistical tests.p = 0.411).Initial construct stiffness was 296.0 ± 50.8 N/mm for group CCS, 365.5 ± 111.1 N/mm for group RST, and 311.3 ± 80.5 N/mm for group RSV, without significant differences among the groups (p ≤ 0.033). The differences between RST and RSV were significant for total displacement (p = 0.020) and with a trend toward significance for torsional displacement (p = 0.061). For gap angle, the outcome measures were homogeneously distributed among the three groups (p = 0.972).The outcome measures for the parameters of interest analyzed over the four time points after 2000, 4000, 6000, 8000, and 10,000 cycles are summarized in Table p ≤ 0.040), with no further significances among the groups (p ≥ 0.140). For the criterion 5° gap angle, the values were without significant differences among the groups, p = 0.180. Earliest failure unexpectedly occurred by 5° torsional displacement of the fragments relative to each other. The failure modes were predominantly expressed by fracturing of the ilium in between the anterior inferior iliac spine and anterior superior iliac spine.The numbers of cycles and the corresponding peak load at the different failure criteria are summarized in Table Group CCS demonstrated considerably higher stability in terms of fracture and torsional displacement compared to group RSTAlthough group CCS revealed comparable results to RSV, it was associated with higher stability regarding cycles to predefined failure criteriaGroup RST was associated with the highest interfragmentary movements and earliest cycles to failure compared to RSV and CCSThe aim of this biomechanical study was to assess the stability of cannulated compression headless screws used for fixation of SPRF. Advances on this topic have the potential to reduce the reported high number of implant failures in the literature, while supporting minimally invasive procedures and possibly providing a new implant with greater stability. Outcome measures of the investigated relative interfragmentary movements lead to the following three primary concluding statements.According to the AO surgery references, either a fully threaded 3.5 mm/4.5 mm cortical screw, or a 6.5/ 7.3 mm cancellous screw without a washer are recommended for SPRF fixation . A washeOpen reduction and internal fixation of pubic rami fractures using plates are known to result in greater blood loss due to the requirement of wide surgical exposure and can result in severe complications such as injuries to the femoral artery, vena or nerve . AnotherGenerally, while using only a single screw, there is always the possibility of fragment rotation around its axis. This seems to be especially present in the smooth, threadless section of a partially threaded screw. In the case of a fully threaded screw, such as the one used in group RSV, intuitively, the rotation should be reduced. This postulation corroborates our finding, since there was a higher rotation in the RST group, yet the differences could not be statistically substantiated as we found no difference between a fully threaded screw and a partially threaded screw.A biomechanical cadaver study comparing the stability of two 3.5 mm fully threaded screws vs. one 7.3 mm partial threaded screw treating SPRF also presented comparable results . On the Regarding the entry point, it has been proven that an antegrade screw placement is less likely to result in fixation failure, when compared to retropubic screw fixation . HoweverTo address the high rate of reported implant related failure, the authors propose a new implant using secure and minimally invasive well-established techniques to stabilize pubic rami fractures by CCHS. There are known predictors of implant failure such as increased patients age and increased patients BMI, distance and decreased distance from the symphysis have been reported . IncreasFurther studies on cadaveric bones should be undertaken in the next step to verify if the findings are reproducible in human bones. If the results are comparable, the transition to clinical studies could then be made.Our results demonstrated a comparable or superior result of the CCHS versus current standard minimally invasive treatment options. However, no screw migration was observed in any of the tests, as seen clinically. The artificial bone seems to have limitations in this regard. Therefore, the used specimen model of artificial bone is the main limitation in this study. However, the authors have performed this first-step investigation because of its novel approach and no available data for comparison. It is further known that artificial bones grant standardized and comparable sample groups, which can overpower the variations in bone quality in human cadaveric specimens and are more cost-effective –31. SyntAdditional biomechanical studies should be performed using human cadaveric bones and larger sample sizes for a better understanding of CCHS and their potential for the minimal invasive treatment of SPFR.The CCHS fixation, introduced in this study, presented predominantly superior stability to the standard surgical treatment and could therefore be a possible alternative implant for retrograde SPRF screw fixation, whereas partially threaded screws in group RST were associated with inferior biomechanical stability.