People on HIV treatment with undetectable virus cannot transmit HIV sexually . However, the science of treatment-as-prevention (TasP) may not be widely understood by people with and without HIV who could benefit from this information. We systematically reviewed the global literature on knowledge and attitudes related to TasP and interventions providing TasP or U = U information. We included studies of providers, patients, and communities from all regions of the world, published 2008–2020. We screened 885 papers and abstracts and identified 72 for inclusion. Studies in high-income settings reported high awareness of TasP but gaps in knowledge about the likelihood of transmission with undetectable HIV. Greater knowledge was associated with more positive attitudes towards TasP. Extant literature shows low awareness of TasP in Africa where 2 in 3 people with HIV live. The emerging evidence on interventions delivering information on TasP suggests beneficial impacts on knowledge, stigma, HIV testing, and viral suppression.Review was pre-registered at PROSPERO: CRD42020153725The online version contains supplementary material available at 10.1007/s10461-021-03296-8. Two decades ago, there was increasing recognition that plasma HIV viremia was highly associated with the risk of HIV transmission , and manDespite the impact of TasP on global HIV policy, information on TasP has been slower to disseminate to people with HIV and people at risk for HIV who might benefit from understanding the prevention benefits of ART . In 2016To inform policy on TasP/U = U information dissemination, we undertook a systematic review of the global literature on beliefs about TasP/U = U. We structured our review around three questions. (1) What are current levels of awareness and knowledge of TasP/U = U among clinical providers, people with HIV in clinical care, and lay community members including people with and without HIV? (2) What are current attitudes regarding TasP/U = U and acceptability of TasP as a prevention strategy in these different populations? (3) What is the impact of interventions disseminating information on TasP/U = U on knowledge, attitudes, behaviors, and health outcomes?https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020153725).We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA) guidelines in preparing this review . We deveStudies were eligible for inclusion if they were empirical studies presenting analyses of data in any of five domains pertaining to TasP/U = U: (1) awareness, (2) knowledge, (3) attitudes, (4) acceptability, and (5) impact. The impact domain included studies that presented data evaluating the effects of interventions providing accurate information regarding TasP or U = U. We included both quantitative and qualitative studies.Our domains focused on theoretical steps leading from new information to behavioral responses, as described in the marketing , 21, heaWe excluded studies that did not analyze and present data, such as opinion pieces, position statements, reviews, guidelines or recommendations, and fact sheets. We excluded studies in languages other than English. We also limited our focus to studies on the prevention of sexual transmission given current gaps in the science on TasP for injecting drug use. For studies of “impact”, we excluded studies where sharing information on TasP/U = U was not a central component of the intervention as well as studies that did not have a control group enabling inferences on causal impact.We included studies published in English between January 1st, 2008 and October 18th, 2020. Studies were included if they were published in a peer-reviewed journal indexed on PubMed, if they were presented at one of three major HIV conferences , or if they were published as a working paper or report available on Google Scholar.We searched PubMed for articles using the following terms in the title or abstract:  AND ("HIV" OR "HIV/AIDS" OR "Human Immunodeficiency Virus"). We then searched conference abstract books for CROI (2014–2020), IAS (2010–2020), and AIDS (2018–2020) using text search functions with the same keywords. We additionally included studies referred to us through personal communication, which were identified on Google Scholar but may not have been indexed in PubMed. After our initial search returned few studies evaluating the impact of TasP/U = U interventions, we reviewed the bibliographies of those studies in the impact domain to identify others we may have missed. We also used Google Scholar’s “cited by” function to identify other, more recent studies that cited these impact studies.In a first screening step, we reviewed titles and abstracts and flagged studies for further review. In a second screening step, we assessed full text manuscripts with respect to inclusion and exclusion criteria and identified the studies to be included in the review. At each step, two authors  conducted the initial screening independently and a third author (JB) reviewed final selections and resolved disagreements. Duplicate records were eliminated. Where a study was presented at a conference and later published, we included just the published paper unless the data presented differed substantially. The study selection process was documented in accordance with the PRISMA-P reporting checklist.For included studies, we extracted the following information into a Google sheet database: year(s) when data were collected, location of study, study population, sampling strategy, sample size, sample characteristics, study methods, and key results. To enable standardized reporting, studies were coded according to domain ; study population ; and region, i.e. North America/Europe, Africa, or other . Region groupings were decided a priori based on income levels and HIV epidemiology; specific countries were also recorded for all studies. In coding study population groups, we defined providers as anyone involved professionally in clinical care for people with HIV or in HIV-related policy; we defined patients as people living with HIV who were identified through involvement in HIV care; and we defined community as people with or without HIV identified in non-clinical settings. Information on specific key populations  was extracted based on descriptors used by study authors and was not coded according to pre-assigned categories. Some studies encompassed more than one domain, population, or region. For studies on impact, we additionally extracted data on the intervention and its comparators and outcomes, following the Population-Intervention-Comparison-Outcome (PICO) framework . Risk ofOur analysis proceeded in three stages. First, we assessed the coverage of the extant literature across domains, population groups, and regions in order to identify gaps. Second, we summarized quantitative measures within each of the key domains, to the extent that comparisons could be made across studies. Third, we assessed for key themes within each domain, drawing on both quantitative and qualitative studies. We did not attempt quantitative synthesis of the data into single metrics due to the diversity of study methods and lack of direct comparability of measures.The study involved no human subjects, and ethics review was not required.Our search strategy identified 886 studies, of which 677 were from PubMed and 209 were from other sources including conference abstract books, reference searches, and personal communication Fig. . After rTable S1. The number of published studies on TasP/U = U beliefs has increased in recent years , just 51% were aware that condomless sex does not lead to HIV transmission in the setting of viral suppression . In a 20Provider knowledge gaps may lead to ineffective communication on TasP. For example, a study in Kenya interviewed both health providers and HIV-negative members of mixed status couples using PrEP in 2017–2018. Heath providers were aware of TasP/U = U but reported incomplete knowledge and inconsistent beliefs: for example, some believed—inaccurately—that U = U only worked with consistent condom use. In turn, members of mixed HIV status couples reported they were informed about U = U by the providers, but that they did not believe the message .Providers reported mixed attitudes about TasP, although most were supportive. In a 2014 international survey of health providers, 17% perceived that other providers were opposed to TasP and 37% were unsure . Some prIn a 2017 U.S. survey, 76% of providers commonly or always recommended condoms to patients with viral suppression, and just 3% of medical providers agreed that condomless sex in the setting of full viral suppression with good adherence could be recommended as “settled science” . In MalaAcceptability of TasP among providers has increased over time as evidence of clinical benefits of early ART and the science of U = U have become clear. In 2012, prior to the START  and TEMPWhile a large number of studies have documented rising knowledge and acceptability of TasP/U = U, very few studies have rigorously evaluated the impact of disseminating TasP information on behaviors and clinical outcomes. Table Kalichman et al.  tested aBuilding on the 2011 study, Kalichman et al.  evaluateTwo studies conducted in sub-Saharan Africa suggest that “light touch” messaging on U = U can also motivate care-seeking behaviors, highlighting the importance of information itself. Smith et al.  conducteWhereas the above studies provided information on TasP at the individual-level, the full benefits of TasP may only be realized once other community members (including prospective sex partners) are aware that ART leading to viral suppression eliminates transmission risk. Comfort disclosing one’s status and discussing TasP may depend on potential partners’ knowledge of TasP. Further, community beliefs may shape care-seeking behaviors among those who think that they might have HIV but fear discrimination from potential sex partners. Community-level awareness of TasP or U = U could shift perceptions of someone using ART from being a high-risk partner to a low- or no-risk partner, leading to greater HIV testing and ART uptake.To assess this pathway, Derksen et al.  conducteMore than 20 years after the risk of HIV transmission was directly correlated with plasma viremia , a decadFirst, awareness regarding TasP/U = U has increased over time, but in-depth knowledge and belief in the scientific evidence remains uneven. Information on TasP/U = U has diffused fastest among MSM populations in Europe, Asia, Oceania, and the Americas, but disbelief is still widespread in these communities, particularly among people who are HIV-negative. Large gaps in knowledge persist in Africa where 2 out of every 3 PLHIV reside. Second, once people believed the science on TasP/U = U, acceptability of TasP was generally high. Among PLHIV, U = U was viewed as enabling a positive self-image and reduced internalized stigma. Among people without HIV, not trusting partner adherence was a barrier to acceptability of TasP. Third, interventions disseminating TasP/U = U information have had beneficial impacts on HIV testing, adherence, viral suppression, and stigma reduction, without leading to increased STI incidence. Fourth, not all health providers are well-informed about TasP/U = U and some have judgmental views which make them unwilling to share information about TasP with their patients.Out of the 72 studies identified in our review, only 17 studies were conducted in Africa and just 5 of these were quantitative studies with population-representative samples. Further research on population knowledge and attitudes related to TasP will be important to guide the implementation of U = U interventions in sub-Saharan Africa. We also found only 4 rigorous studies on the effect of TasP/U = U messaging globally. More research is needed to assess the impact of different types of TasP/U = U interventions (intensive vs. light-touch), implemented in different contexts (from the counselling room to the street-corner), delivered by different people (nurses vs. pastors vs. peer educators), in populations with different sexual behaviors, in different health-systems contexts, in cultures with different mental models of disease transmission, implemented at different levels , targeting different theoretical pathways to impact  in addition to qualitative endorsements of statements about TasP/U = U , 41, 42.Fifth, most existing studies of impact delivered TasP/U = U messaging alongside other interventions such as enhanced adherence counseling , 86 or eThe science of TasP has transformed global HIV policy, with countries worldwide embracing test-and-treat and many now engaging in public information campaigns about TasP/U = U. Our review finds that there remain large gaps in lay knowledge about TasP, particularly in Africa and particularly among people without HIV. Our findings also indicate that disseminating information on TasP/U = U could increase HIV testing, reduce stigma, and improve HIV treatment outcomes, leading to better health for PLHIV and lower risk of transmission to others.Supplementary file1 (XLSX 528 KB)Below is the link to the electronic supplementary material.


On the front lines of climate change, glacier termini play crucial roles in linking glaciers and downstream ecosystems during glacier retreat. However, we lack a clear understanding of biological processes that occur in surface and basal ice at glacier termini.Here, we studied the bacterial communities in surface ice and basal ice (the bottom layer) of a glacier terminus in the Yangtze River Source, Qinghai–Tibet Plateau.Surface and basal ice harbored distinct bacterial communities but shared some core taxa. Surface ice communities had a higher α-diversity than those in basal ice and were dominated by Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Cyanobacteria while basal ice was dominated by Firmicutes and Proteobacteria. The bacterial communities were also substantially different in functional potential. Genes associated with functional categories of cellular processes and metabolism were significantly enriched in surface ice, while genes connected to environmental information processing were enriched in basal ice. In terms of biogeochemical cycles of carbon, nitrogen, phosphorus, and sulfur, bacterial communities in surface ice were enriched for genes connected to aerobic carbon fixation, aerobic respiration, denitrification, nitrogen assimilation, nitrogen mineralization, sulfur mineralization, alkaline phosphatase, and polyphosphate kinase. In contrast, bacterial communities in basal ice were enriched for genes involved in anaerobic carbon fixation, fermentation, nitrate reduction, 2-aminoethylphosphonic acid pathway, G3P transporter, glycerophosphodiester phosphodiesterase, and exopolyphosphatase. Structural equation modeling showed that total nitrogen and environmental carbon:phosphorus were positively while environmental nitrogen:phosphorus was negatively associated with taxonomic β-diversity which itself was strongly associated with functional β-diversity of bacterial communities.This study furthers our understanding of biogeochemical cycling of the mountain cryosphere by revealing the genetic potential of the bacterial communities in surface and basal ice at the glacier terminus, providing new insights into glacial ecology as well as the influences of glacier retreat on downstream systems.The online version contains supplementary material available at 10.1186/s40793-022-00408-2. Covering approximately 10% of land surface, glaciers contain 75% of the Earth’s freshwater as ice and are among the least explored environments on Earth , 64. GlaIn glacier termini, surface ice and basal ice exhibit distinct physicochemical environments . The surWe conducted the study in a glacier terminus on the Qinghai–Tibet Plateau (QTP). As the “Water Tower of Asia” and the Third Pole, QTP harbors the largest number of glaciers outside polar regions . However2, the Donkemadi Glacier has an altitude between 5420 to 5919 m, an annual precipitation of 680 mm, and an annual air temperature of − 8.6 ℃ [Fieldwork was conducted at the terminus of Dongkemadi Glacier , which is located in the headwaters of the Yangtze River in the central QTP Fig. . Coverin − 8.6 ℃ , 15. Sin − 8.6 ℃ , SI sampIce samples were melted under room temperature at the field station. Microbial samples were collected by filtering 200 mL meltwater onto a 0.2-μm polycarbonate membrane filter . Filters were frozen in liquid nitrogen immediately and stored at − 80 ℃ in the lab until DNA extraction. pH and conductivity were measured using a multiparameter instrument . For each sample site, three 1-L acid cleaned bottles were filled with meltwater and transported to the laboratory for chemical analyses. Dissolved organic carbon (DOC) was analyzed using filtered (using pre-combusted GF/F filters) meltwater on a Shimadzu TOC Analyzer . Total nitrogen (TN) and total phosphorus (TP) were measured using the unfiltered water. TN was measured using ion chromatography after persulfate oxidation. TP was measured by using the ascorbate acid colorimetric method after oxidation. The details of these methods and findings are summarized elsewhere .DNA from the filter samples was extracted using the DNeasy PowerSoil Kit  following the manufacturer’s protocols. Meanwhile, three blank controls were conducted using the same processes for the samples in order to eliminate the laboratory contamination. The forward primer 343F 5′-TACGGRAGGCAGCAG-3′ and reverse primer 798R 5′-AGGGTATCTAATCCT-3′ [single_rarefaction.py script. The alpha diversity indices, including Chao 1, observed OTUs, Simpson, and Shannon, were calculated using the alpha_diversity.py script in QIIME 1.9.1 [Raw sequence data were first preprocessed to detect and cut off ambiguous bases and low-quality sequences  using Trimmomatic (version 0.35) . After tME 1.9.1 .https://www.genome.jp/kegg/) were clustered to different pathway levels (levels 1–3) using the predicted metagenomes. The KOs associated with the biogeochemical cycles of carbon, nitrogen, sulfur, and phosphorus were further extracted  . We calchttps://itol.embl.de/). Then, non-metric multidimensional scaling was used to reveal the differences of taxonomic and functional composition of bacterial communities between surface ice and basal ice based on the relative abundance of OTUs and KOs, using Vegan package 2.5-7 [We compared taxonomic structure and functional potential of bacterial communities in surface ice and basal ice. The phylogenetic tree of all the detected 464 OTUs was built using iTOL , Firmicutes (90 OTUs), Bacteroidetes (84 OTUs), Actinobacteria (40 OTUs), and Cyanobacteria (18 OTUs) Fig. . The rem60 OTUs, Surface ice had significantly higher alpha diversity than basal ice Fig. a. The avIn surface ice, the most abundant phylum was Proteobacteria (33.6%), followed by Firmicutes (26.7%), Bacteroidetes (16.6%), Cyanobacteria 15.4%), and Actinobacteria (5.6%) . In general, bacterial communities in surface ice and basal ice had significantly different functional compositions Fig. . NotablyA combination of normalized marker genes  in surface ice and basal ice Fig. . Based oIn glacier termini, surface ice and basal ice are two distinct environments with substantially different physicochemical properties . Our datBacillus, Lactobacillus, Streptococcus, Lactococcus, Enterococcus, and Klebsiella  and imbalanced , 51, 52.Upstream catchments export biotic and abiotic materials from various glacial and non-glacial sources to downstream aquatic ecosystems, substantially influencing ecosystem structure and function of glacier-fed streams and lakes , 59, 70.Additional file 1. List of the KEGG orthologs associated with carbon, nitrogen, phosphorus, and sulfur cycles. List of OTUs with relative abundance above 1% in surface ice and/or basial ice.Additional file 2. OTU table of the sample sites.


In this review, we summarize various site-selective reactions mediated by molecular containers. The emphasis is on those reactions that give different product distributions on the potential reactive sites inside the containers than they do outside, free in solution. Specific cases include site-selective cycloaddition and addition of arenes, reduction of epoxides, α,β-unsaturated aldehydes, azides, halides and alkenes, oxidation of remote C–H bonds and alkenes, and substitution reactions involving ring-opening cyclization of epoxides, nucleophilic substitution of allylic chlorides, and hydrolysis reactions. The product selectivity is interpreted as the consequence of the space shape and environment inside the container. The containers include supramolecular structures self-assembled through metal/ligand interactions or hydrogen bonding and open-ended covalent structures such as cyclodextrins and cavitands. Challenges and prospects for the future are also provided. To run reactions with discriminate control over product selectivity represents one of the huge challenges in organic synthetic chemistry , among wAfter decades of rapid development, supramolecular chemistry has won two times for Nobel Prizes and already became one of the most important fields in modern chemistry –15. It iA was used, the Diels–Alder reaction of 9-hydroxymethylanthracene (1) and N-cyclohexylphthalimide (2) went smoothly at 80 °C for 5 hours with near quantitative yield  almost quantitatively affording the 9,10-adduct 6 at room temperature for 5 hours. Control experiments proved the promoting effect of the hydrophobic pocket of B. The origin of the catalytic behavior of the bowl host B can be explained by to two main aspects. Firstly, the bowl host B possesses an open cavity that facilitates rapid binding and dissociation of the guests. Secondly and more importantly, before the reaction, the anthracene moiety stacks onto the planar triazine of B through π–π stacking and possible charge-transfer interaction between each other, which stabilizes the complex. However, after the reaction, the framework of the product is bent at the 9,10-position, which undermines the host–guest stacking interaction. The complex of B and the product is hence destabilized, resulting in the replacement by incoming reactants. This beautiful pioneering work showed elegant examples of how the designed and synthetic molecular containers could mediate and even control the site-selectivity of organic reactions.Cycloaddition reactions have long been applied to molecular container-mediated enzyme-mimicking transformations ,31–33, ave yield . Only th product . This kiturnover . Only 102 and 2,3-substituted naphthalene 7 mediated by cage host A mentioned above , and after being heated at 100 °C for 8 hours, the site-selective product 8 was obtained with moderate yield. No other side reactions were observed, and the moderate yield resulted from the partial sublimation of the reactants. The reaction proceeded site-selectively at the unsubstituted ring of the 2,3-substituted naphthalene, and produced stereoselectively the syn isomer 8, which was determined and confirmed by multiple characterization methods including NMR, mass, and X-ray crystallographic analysis. The reaction did not take place without the cage host A. Alkyl substituents at the C2 and C3 position of naphthalene were crucial to this reaction. Control experiments upon the substituent effect indicated that it was the steric, not the electronic factor, that ruled the reactivity and selectivity. The electron-donating alkyl groups should have facilitated reaction at the substituted ring, in contrast, the reaction occurred at the unsubstituted ring. This unusual site-selectivity can be explained by the preorganization of the reactants within the cage host A. In the confined space of the cavity, the orientation of the substrates was fixed presumably with the unsubstituted ring of naphthalene subject to the double bond of 2. This work showed the remarkable function of molecular containers to override natural reactivity and produce unusual site-selective products. Intriguingly, upon irradiation, a site- and stereoselective [2 + 2]-photoaddition between 2 and 7 took place smoothly, giving rise to the syn isomer of the 5,6-adduct 9. In a following work, the authors reported similar site-selective Diels–Alder reactions between 2 and inert aromatics including aceanthrylene and 1H-cyclopenta[l]phenanthrene . Gi. Gi2 andanthrene .A, the authors realized a highly site-selective radical addition reaction of o-quinone 10 and substituted toluene 11, giving rise to the unusual 1,4-adduct 15 . Utilizing the similar molecular container as the noncovalent protective group, the Pitchumani and Srinivasan group also reported that the reduction of coumarin (18) by sodium borohydride could be site-selectively induced in the presence of β-cyclodextrin C  in aqueous solution with a cavitand host as the protecting group for one of the azide sites  ineactions . The feeeactions . Before E and F was reported later  to make sure the reactions occur under confinement in the host. When using a rigidified host F, the secondary alkyl dibromide 22 was transformed to the monoreduced product 23 with high site-selectivity, which benefited from the high KA value (≈1.5 × 105 M−1) (3SiH) using the similar strategy [Another site-selective radical monoreduction of dihalides mediated by water-soluble cavitand hosts ed later  62]. Ph. PhE andcavitand . Experim105 M−1) . This wostrategy .4L612− cage host G, which had a relatively larger size with pyrene-walled ligands -bounded porphyrin module carrying four β-cyclodextrin units at the end. Once the two parts were mixed in the reaction system, the two p-tert-butylphenyl groups of the substrate 27 were recognized by the β-cyclodextrin and anchored through the host–guest binding. At this stage, the steroid core exposed the 6-position C–H bond to the metalloporphyrin unit catalytic center and gave rise to the site-selective 6-hydroxy derivative, which was then hydrolyzed to the final site-selective product 28. The series of work showed that the powerful molecular container could be used as the anchoring group in the template catalysis. Subsequent reports by other groups illustrated that apart from molecular containers, other designed moieties could also be used to anchor the substrate through hydrogen-bonding interactions . Us. UsJ (74o 3.8:1) . In thisD and E (44 showed rapidly exchanging and folded J-shape conformations, which exposed each ester group in turn to the aqueous solution. Addition of base would result in the hydrolysis of one ester group to the corresponding carboxylic acid. Product distributions indicated a two- to four-fold relative decrease in the hydrolysis rate constant of the second ester caused by the confined space in the cavitand, which enhanced the selectivity of the monoester product 45. Similarly, the monohydrolysis of α,ω-diisocyanate 46 was achieved using the water-soluble cavitand D (D and protected from further hydrolysis. The monoamine product 47 further underwent intramolecular cyclization facilitated by the confinement of the cavitand and produced cyclized urea product 48. In a following work (49 showed rapid tumbling conformation in the cavity of the cavitand host E on the NMR timescale. DMSO was introduced as a co-solvent to promote the SN2-type reaction, and the dibromide 49 was smoothly hydrolyzed site-selectively to the monohydroxy product 50, which was fixed in an unsymmetrical manner in the cavitand. The bromide terminal was hence protected deep within the cavity of the cavitand host E from further hydrolysis, despite the addition of 10 equivalents DMSO after 2 days or even after one month. The experiment without the cavitand host produced a mixture of different products, and the dibromide was fully converted to the dihydroxy product after prolonging the reaction time or increasing DMSO concentration, without the detection of any monohydrolyzed product. This work further demonstrated the striking ability of the water-soluble cavitand to mediate site-selective reactions.The Rebek group later reported a series of site-selective monohydrolyses of α,ω-difunctional compounds using deep water-soluble cavitands D and E  76–78].–78.D andvitand D  77]. Th. ThD anding work  [78], thTo summarize, we have reviewed various site-selective reactions mediated by molecular containers, which have drawn much attention in the past years and shown broad prospects in the future. The supramolecular cavity and its constrained microenvironment resemble the active site of natural enzymes, where the guest substrate is encapsulated and positioned with a specific fixed orientation and conformation through various noncovalent interactions, giving rise to discriminate control over product selectivity. In some cases, the molecular container is considered as a noncovalent protective group that prevents potential reactive sites from reacting with external reagents; in other cases, the molecular container acts as the anchoring template and fixes the substrate with a certain conformation, exposing one reactive site to the catalyst center and producing site-selective products. Even though molecular containers have been proved to be powerful tools in inducing reaction selectivity, there are still some restrictions that should be considered. For example, the substrate scopes of these methodologies are generally limited; sometimes, a near quantitative amount of the supramolecular host is required and the synthesis of macromolecular hosts is sometimes complicated. Future research should focus on those limitations as well as developing diverse catalysis systems that would induce controllable site-selectivity.


Rb1 is a regulator of cell cycle progression and genomic stability. This review focuses on post-translational modifications, their effect on Rb1 interactors, and their role in intracellular signaling in the context of cancer development. Finally, we highlight potential approaches to harness these post-translational modifications to design novel effective anticancer therapies.The retinoblastoma protein (Rb1) is a prototypical tumor suppressor protein whose role was described more than 40 years ago. Together with p107  and p130 , the Rb1 belongs to a family of structurally and functionally similar proteins that inhibits cell cycle progression. Given the central role of Rb1 in regulating proliferation, its expression or function is altered in most types of cancer. One of the mechanisms underlying Rb-mediated cell cycle inhibition is the binding and repression of E2F transcription factors, and these processes are dependent on Rb1 phosphorylation status. However, recent work shows that Rb1 is a convergent point of many pathways and thus the regulation of its function through post-translational modifications is more complex than initially expected. Moreover, depending on the context, downstream signaling can be both E2F-dependent and -independent. This review seeks to summarize the most recent research on Rb1 function and regulation and discuss potential avenues for the design of novel cancer therapies. Rb1 locus is located on the q arm of chromosome 13, and the presence of genetic rearrangements in this region in retinoblastoma tumors was one of the crucial pieces of evidence for the identification of Rb1 as a key player in the development of such tumors [The discovery of the Retinoblastoma-associated protein 1 (Rb1) in the late 1970s was an important milestone in understanding cancer development ,3. Untilh tumors ,7.Rb1 comprises 928 amino acids, has a molecular weight of 106 kDa, and belongs to a family of structurally related molecules called pocket domain proteins. The family consists of Rb1 and its two paralogs Rbl1 (p107) and Rbl2 (p130), which share similar domain organization, although several differences make Rbl1 and Rbl2 more related to each other than to Rb1 [The product of Rb1 protein has several domains that are crucial for its function and interactions. In the past, the central pocket domain (residues 379—792) and C-terminal domain (residues 792—926) were considered the most important parts for Rb1 function , namely In this review, we describe the canonical and non-canonical functions of Rb1, its alteration in human cancers, and the post-translational modifications that are crucial for its regulation. Finally, we discuss how Rb1 post-translation modifications could be harnessed to design novel anticancer therapies.To date, more than 300 Rb1 interactors have been identified, thus indicating that this protein is involved in multiple signaling complexes . AlthougThe key event for the regulation of E2F1-dependent downstream signaling is the binding of Rb1 to E2F1. This interaction and its consequences have been studied extensively and the mechanisms involved are well characterized.Cyclin A, Cyclin E and cdc25, which ultimately leads to cell cycle arrest and inhibition of cell proliferation [Initial binding analyses and structural experiments determined two structural features that delineate the binding of Rb1 to E2F1, namely the central pocket and C-terminal domains ,18,19 FA. The ceferation ,24,25).Structure and interaction surfaces are regulated by phosphorylation by CKDs. There are two main regions on Rb1 that are phosphorylated by CDKs—central pocket domain and C-terminal domain. Phosphorylation of residues within these regions  leads to disruption of Rb1 with E2F1 interaction and transcriptional activation of cell cycle regulating genes (More detailed description of these phosphorylations is in following sections).Rb1-mediated inhibition of E2F transcriptional activity is mediated by two distinct molecular mechanisms. First, Rb1 binding to the transactivation domain of E2F blocks the recruitment of general transcription factors and histone acetylases that are necessary for transcription initiation ,27. SecoAfter the recruitment of HDACs to E2F-target promoters, the deacetylases act on the surrounding histone tail residues, thus repressing E2F-mediated transcription. Moreover, this repression is dependent on Rb1 and also on HDAC activity, as trichostatin A treatment (HDACi) impairs this inhibition ,30. ReceAn interesting expansion of the Rb1 function is the regulation of silencing of repetitive DNA sequences, such as endogenous retroviruses or LINE-1 elements. These elements bind E2F1, which results in cell cycle-independent recruitment of Rb1. Epigenetic silencing of these elements is dependent on H3K27 trimethylation and it is markedly reduced in cells where Rb1 recruitment is impaired. The underlying mechanism is Rb1-dependent recruitment of EZH2 to the repetitive elements. Moreover, E2F1 binding-deficient Rb1 was still able to associate with EZH2, however, there was no deposition of methylation to the repetitive sequences, which confirms that maintenance of epigenetic silencing of the repetitive sequence is dependent on the formation of the E2F1–Rb1–EZH2 tricomplex .The interaction of Rb1 with E2F is regulated by either post-translational modification of Rb1, which is discussed in the next section, or by binding with certain cellular and viral proteins. Among the latter, viral proteins, such as adenovirus (Ad) E1A, human papillomavirus (HPV) E7, and simian virus 40 (SV40) large T antigen (LTa), have been characterized in most detail ,42,43,44In addition to E2F1, Rb1 can bind other members of the E2F family, thereby adding further complexity to the role of this protein. E2F2, E2F3, E2F4 and E2F5 members contain Rb1-binding domains similar to E2F1 and interact with Rb1 ,49. The The mechanism by which Rb1 regulates E2F-mediated transcription has been extensively studied; however, Rb1 binds to multiple other cellular proteins and alters their function independently of E2F, thereby affecting cell cycle progression, differentiation and transcription.The E2F-independent role of Rb1 is well established in the context of lineage commitment and terminal differentiation of different cell types. Rb1 interacts with several transcription factors, such as MyoD, Runx2, C/EBP, NF-IL6, GATA1 and Pax8, and acts as a transcriptional co-activator. This interaction subsequently leads to stimulation of lineage-specific gene transcription and progenitor differentiation into mature tissue-specific cells, such as adipocytes, myocytes, hematopoietic cells and osteocytes ,55,56,57Another important transcription factor bound by Rb1 is p65; however, there is no definitive consensus on the nature of this interaction. An initial study conducted on osteosarcoma cells showed that Rb1 binds and stimulates transcriptional activity of p65, which leads to an increased expression of cyclin D1 . On the Another process directly associated with transcription control is an epigenetic modification of chromatin. As described above, Rb1 can recruit chromatin-modifying enzymes to E2F-target gene promoters and thus alter E2F-stimulated transcription. However, some reports indicate that Rb1 facilitates the function of several histone modifiers also in an E2F-independent manner. One such enzyme is histone demethylase KDM5a (RBP2), which demethylates tri- and di-methylated H3K4 . The intSkp2 ubiquitination complex and its stabilization. Increased levels of p27 subsequently result in the inhibition of CDK activity and cell cycle arrest [In addition to the CDK–Rb1–E2F axis, Rb1 regulates cell cycle progression via several E2F-independent mechanisms. One such mechanism is the interaction with ubiquitin ligase complexes SCF and APC/C, which facilitate the transition between phases of the cell cycle ,66. Rb1 e arrest . The APCe arrest . Rb1 inte arrest ,69  or more often through functional inactivation by CDK-stimulated hyperphosphorylation. In addition to this canonical function, Rb1 is also important for the maintenance of genomic and chromosomal stability. These roles of Rb1 are addressed in the following sections.Rb1 locus was associated with the development of various types of cancer, including sarcoma, lung and ovarian cancer [Rb1 is altered  in 7% of all samples. However, mutational frequencies vary between tumor types, with bladder cancer and sarcoma samples having the highest proportion of Rb1 mutations (20–25%), followed by prostate cancer, melanoma, and liver and brain cancer (10–15%). Most of these mutations (88%) are classified as driver mutations, which result in the absence of functional Rb1 protein . These frequencies are consistent with Rb1 being a negative regulator of the cell cycle. Moreover, 16% of the alterations are missense mutations  repair by all three major DSB repair pathways—homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ). Rb1 localizes to the sites of DNA damage in an E2F1- and ATM (serine-threonine kinase ataxia-telangiectasia mutated)-dependent manner. Mechanistically, ATM-phosphorylates E2F1 at S29, which leads to its recruitment to p-γH2AX foci. Rb1 is localized to these foci via association with E2F1, and Brg1 DNA helicase is subsequently recruited in an Rb1-dependent fashion . RecruitRb1 dosage is strongly associated with the protection of cells from chromosomal alterations and aneuploidy. Full Rb1 deletion leads to a high rate of chromosomal instability, and even deletion of a single copy has a profound effect. Mechanistically, Rb1 recruits Cohesin II to the centromeric regions and thus promotes faithful segregation of sister chromatids [In addition to its direct role in the DSB repair, Rb1 also facilitates genome integrity through stabilizing heterochromatin. As described earlier, Rb1 interacts with and recruits several epigenetic factors that alter chromatin structure. It is now well established that Rb1 plays a crucial role in stabilizing heterochromatin at centromeric and telomeric regions through maintenance of sufficient levels of H4K20 methylation. Rb1-deficient mouse fibroblasts or fibroblasts expressing Rb1 mutated in the LxCxE binding cleft show decondensed chromatin and decreased H4K20 levels specifically at telomeres and pericentric chromatin ,64. Thisromatids ,105. In romatids ,107,108.Finally, yet importantly, some functions of Rb1 can be appreciated only in the context of the cancer microenvironment and interaction with immune cells. Several reports demonstrate the involvement of the E2F1/Rb1 axis in the regulation of antitumor immune response. Most of these focused on the outcome of systemic CDK4/6 inhibition, which was evaluated mainly as the effect on immune cell proliferation and recruitment ,111,112.Similarly, and as mentioned above, it was shown that Rb1 participates in regulating PD-L1 expression; however, this function is paradoxical. A recent study revealed that CDK4/6-mediated phosphorylation of Rb1 on residues S249 and T252 increases its affinity to p65 and inhibits its transcriptional activity. Since one of the p65 targets is PD-L1, this inhibition leads to decreased PD-L1 expression and subsequent impairment of immune evasion .Moreover, Rb1 binds to EZH2 and alters the epigenetic modification status of certain sequences . Such inGiven that Rb1 was described as a prototypical tumor suppressor more than 40 years ago, there is a huge amount of evidence regarding its post-translational modifications. According to phosphositeplus.org, there are 105 modified residues in Rb1. Phosphorylation (61 residues) and ubiquitination (29 residues) are the most common modifications of this protein, while others  have received less attention. The following chapter describes these post-translational modifications, along with the upstream signaling and downstream significance , Table 1The prototypical and most widely studied post-translational modification of Rb1 is phosphorylation. It was the first modification identified to be crucially important for Rb1 function as a tumor suppressor . To dateSince E2F1 was considered the most important Rb1 interaction partner, the effect of phosphorylation of these residues on the Rb1–E2F1 interaction was studied mainly with a focus on the interaction with E2F1, leaving the possibility that some of the phosphorylations may be more relevant for alternative binding partners. Initially, it was reported that phosphorylation of S788 and S795 directly inhibits the binding of Rb1 to E2F1, whereas phosphorylation of T821 and T826 facilitates the closing of the Rb1 structure as a result of increased binding of its C-terminal and central pocket domain . FurtherSince Rb1 is a major regulator of the cell cycle, it integrates signals from stress response pathways such as DNA damage and environmental stresses. It was shown that S612 is phosphorylated by Chk1/2 in response to DNA damage and that this phosphorylation increases its affinity towards E2F1 . SimilarFurthermore, Rb1 is a direct target of the stress-activated kinase p38α, which integrates stress signaling beyond DNA damage. p38α is a master regulator of stress signaling pathways  and, indIn addition to p38α, other reports showed that S249/T252 can be also phosphorylated by CDKs, which results in altered binding with other Rb1 interaction partners such as p65 and HDAC5 ,61. ThisAnother well-described p38α-directed Rb1 phosphorylation site is S567. This phosphorylation is triggered by DNA damage and it results in the disassociation of Rb1 from E2F1, increased interaction with HDM2 ubiquitin ligase, Rb1 poly-ubiquitination and subsequent targeting to proteasomal degradation ,130. ThiOf note, the isoform p38γ cooperates with CDKs, regulating entry into the cell cycle. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of Rb1 at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor protects against the chemically induced formation of liver tumors .Rb1 is directly phosphorylated by the mitotic kinase Aurora B at S780. This phosphorylation is important for mediating the role of Aurora B in regulating the postmitotic duplication checkpoint, thus protecting the cells from aneuploidy . These rIn contrast to Rb1 phosphorylation, very little is known about the dephosphorylation of this protein. Two major phosphatases, namely PP1 and PP2A, were described for Rb1 dephosphorylation . AlthougA second phosphatase involved in mediating Rb1 dephosphorylation is PP2A. Two complementary mechanisms of action were proposed for this phosphatase role. On the one hand, PP2A dephosphorylates CDKs, thereby resulting in lower phosphorylation activity towards Rb1. This mechanism is present mainly in normally cycling, unchallenged cells . On the In addition to phosphorylation, various other post-translational modifications, namely acetylation, methylation and sumoylation, modulate Rb1 function. Several high throughput proteomic studies identified six acetylated lysine residues on the Rb1 protein ,199. HowSimilar to acetylation, lysine and arginine methylation also affect the functionality of Rb1. The most studied site of methylation is K810. This conserved residue lies next to an important CDK phosphorylation site, S807/811, thus implying functional interaction between these post-translational modifications. K810 is methylated by the methyltransferase Set7/9 and this methylation prevents CDK binding and subsequent Rb1 phosphorylation. Moreover, this methylation is stimulated after DNA damage and it appears to play a crucial role in cell cycle arrest upon DNA damage, by two mechanisms. The first would be the promotion of canonical binding to E2F1, followed by recruitment of HP1 and repression of cell cycle-promoting genes . The secFinally, Rb1 can be also subjected to SUMOylation. This post-translational modification is blocked by interaction with small viral proteins; however, the significance of this modification has not been addressed in depth ,135.Rb1 is a multifunctional protein that integrates signals from multiple upstream events and plays important roles beyond the regulation of the cell cycle. Rb1 is extensively modified by phosphorylation, acetylation and methylation. Although the interplay between the qualitative and quantitative extent of these modifications is only now starting to be uncovered, a highly complex regulation of the Rb1 function is emerging. It is becoming clear that the modification of the same residue can result in different outcomes depending on other modifications present, and that there are pools or subpopulations of Rb1 molecules that are differentially modified and play different roles. It could be postulated that the downstream functional output of Rb1 is regulated by a “post-translational modification code”. This mechanism of action can be illustrated by the differential output of Rb1–E2F1 interaction in cycling cells and cells undergoing DNA damage. Blocking Rb1 hyperphosphorylation, which would lead to the increased interaction of Rb1 and E2F1, is a favorable outcome on the promoters of cell cycle genes, but it is unfavorable at the pro-apoptotic gene promoters and could lead to insensitivity to DNA damage. Thus, deciphering the “post-translational modification code” of Rb1 might prove crucial to decouple these two signaling events. Moreover, it was shown that some modifications are dominant over others and that they determine the overall output independently of the other ones present.The importance of the Rb1–E2F signaling axis in cancer is well recognized, however, the only clinically approved drugs targeting the pathway are CDK inhibitors. The efficacy of these inhibitors is dependent on the Rb1 status, thus they cannot be used for patients with non-functional Rb1 protein  . On the In summary, the crucial role of Rb1 in cell cycle regulation calls for a deeper understanding of its modifications, which in turn would contribute to the design of targeted compounds of therapeutic interest.


Nares Strait is a major pathway along which multi-year sea ice leaves the Arctic, an ice class that has seen a recent dramatic reduction in extent. The winds that blow along the strait play an important role in modulating this ice export as well as in establishing the Arctic’s largest and most productive polynya, the North Water, that forms at its southern terminus. However, its remote location has limited our knowledge of the winds along the strait. Here we use automatic weather station data from Hans Island, in the middle of the strait, to assess the ability of a set of atmospheric renalyses and analyses with a common lineage but with varying horizontal resolution to represent the variability in the wind field. We find that the flow is highly bidirectional, consistent with topographic channeling, with the highest wind speeds from the north and that a model resolution of ~ 9 km is required to capture the observed variability. The wind field at Hans Island is also found to be representative of variability in the flow along much of Nares Strait. The wind field in such a narrow channel is typically ageostrophic and controlled by the pressure gradient in the along-strait direction2. This channeling is enhanced by the common occurrence of low-level temperature inversions in the region3 that serve to inhibit vertical motion4.Nares Strait is the ~ 600 km long and ~ 40-80 km wide strait that connects the Arctic Ocean’s Lincoln Sea to northern Baffin Bay Fig. . The ste5 and the strait is a major pathway for the export of this important ice class out of the Arctic6. The wind field along Nares Strait with its preference for northerly flow plays an important role in the export1. Ice arches that form most winters along Nares Strait can result in the cessation of this ice transport for months at a time7.The oldest and thickest sea ice in the Arctic is situated to the north of Nares Strait8. With an arch present, ice motion along the strait is inhibited and the northerly flow down Nares Strait, that accelerates in the exit region of Smith Sound, is able to remove sea ice thus contributing to the maintenance of the polynya’s open water9.The presence of these arches also contributes to the largest and most productive polynya in the Arctic, the North Water (NOW) that forms at the southern end of Nares Strait Fig. , in the 7 and that the area of the NOW is increasing10. There is also evidence that anomalous winds play a role in the collapse of these arches11. In addition, the Arctic is undergoing a transition towards a younger12 and thinner13 ice pack that is leading to a loss of multi-year ice from the Arctic14. There has also recently been an increase in both ice area and ice volume fluxes along Nares Strait7 that has been suggested to contribute to the loss of multi-year ice from the Arctic6.There is evidence that the ice arches along Nares Strait are becoming less stableThe remote and data sparse nature of the region implies that there is still considerable uncertainty regarding the characteristics of the wind field that limits our ability to fully understand its role in ice export and its contribution to the formation of the NOW as well as the wind’s role in the changes that are occurring in the region.−115. However observations extending back to the 1860s indicate that long-lived gales, events characterized by northerly surface wind speeds in excess of 20 ms−1, are a common occurrence in the Smith Sound region during the winter months16.Limited wind observations from the Smith Sound region confirm the preference for northerly flow with mean wind speeds on the order of 6–8 ms−118. During the winter of 1871–1872, the United States North Polar Expedition’s ship was trapped in ice at an exposed site in Hall Basin and the science party regularly observed wind speeds in excess of 20 ms−1 with a preference for northerly flow19. In the vicinity of Robeson Channel, meteorological observations were made at Fort Conger from August 1881 to August 1883 during the First International Polar Year by members of the United States Expedition to Lady Franklin Bay20. These observations indicated the preference for bidirectional wind flow, either in the NNE or SSW direction, along Robeson Channel. There were numerous events, with both directionalities, where the wind speeds were in excess of 15 ms−120.Wind observations farther north along Nares Strait are even rarer. In April 2005, an ice camp established along the Kennedy Channel was destroyed by northerly winds in excess of 25 mso49′35'N, 66°27′35'W), jointly claimed by both Canada and Denmark, is situated in the middle of the Kennedy Channel, one of the narrower sections of Nares Strait  clearly represents the narrowness of Nares Strait in the vicinity of Hans Island as well as the steep topography along both sides of the strait. The eERA5 cross-section , ~ 30 km (ERA5) and ~ 9 km (ECOA). The common lineage of the models allows one to control for biases arising from difference in model architecture and parameterizationsThe comparison of cross-sections through the reference and model topographies Fig.  clearly −1 for the ERA5 to 2.18 ms−1 for the ECOA; while the slope of the least squares linear fit increased from 0.39 for the ERA5 to 0.8 for the ECOA. The latter characteristic implies that the eERA5 and the ERA5 have a tendency to underestimate the wind speed during high wind speed events to a greater extent than the ECOA.The comparison for the 10 m wind speed Fig.  shows a This may be related to the inability of these models to represent the open water in Nares Strait and its replacement with land that generally has higher surface roughness Fig. . Neverth1. All the models, to a greater or lesser extent, represent the narrowness of the strait and hence the tendency for the flow to be ageostrophic and controlled by the pressure gradient in the along-strait, i.e. meridional, direction. The composite high speed events . Included is the new fifth generation reanalysis from the ECMWF or ERA5 with a horizontal resolution of ~ 30 km, as well as its ensemble version, eERA5 with a horizontal resolution of ~ 60km21.For this study, the instantaneous 6-hourly 10 m winds and 100 m from these three datasets were interpolated to the location of Hans Island and the data was used at those times for which there are observations. A number of different interpolation schemes were used and the results were independent of scheme. For simplicity, the results shown are for a bilinear interpolation scheme. In total, ~ 550 days worth of data was included in this study. The AWS was situated at a height of approximately 170 m above sea level


Group3) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MBGroup3 stem cells, and a combined anti-autophagy and anti-STAT3 approach impacts the MBGroup3 outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBThe online version contains supplementary material available at 10.1007/s00401-021-02347-7. To strengthen our findings, we also used MBSCs from human MB (hum MBSCs)  to target Elongin C (ELOC), an essential adaptor protein that mediates the assembly of the CRL5-SOCS3 complex, which includes CUL5 . This covel Fig. e (Supp. MYC and CYCLIN D2)  Fig. a (Supp. D2) Fig. b, c  overexpression in P0 CD1 mice cerebella, as previously described [Since MB represents a very heterogeneous family of tumours requiring different therapeutic approaches to obtain reliable results, we decided to test the combination of both WP1066 and CQ in a MBescribed . Mice trIn sum, we can conclude that upon c-MYC/MIZ-1-mediated upregulation of AMBRA1 transcription, this factor enhances both autophagy and STAT3-c-MYC signalling in high-risk MB. Molecular dissection of this axis primed us to demonstrate that acting on both pathways may represent a valid combined approach to counteract high-risk MB growth and spread Fig. h.Group3 stem cells and that its silencing has a strong effect on stem potential, viability and migration. This phenotype is recapitulated only in part by inhibition of autophagy, through a mechanism that remains to be elucidated, this suggesting the involvement of multiple specific regulations mediated by AMBRA1. Indeed, upon AMBRA1 depletion, a dramatic inhibition of STAT3 signalling occurs, through the stabilization of SOCS3, with this confirming that MB stem potential and cell survival also depends in part on AMBRA1 regulation.As previously shown, AMBRA1 is an important factor at the crossroad between autophagy, development and cancer , 50, 82.Group3 cell stem potential and growth, our work identifies a new AMBRA1-c-MYC regulatory axis. On one hand, we propose that c-MYC overexpression induces AMBRA1 transcription in a MIZ-1-dependent manner.By dissecting the molecular details of AMBRA1 transcriptional enhancement, fundamental for MBGroup3, modulating the expression of specific genes that maintain the identity of these tumours and distinguish them from other MB subgroups [Group3 relative to MBSHH. On the other hand, loss of AMBRA1 in MBGroup3 is sufficient to decrease the expression of STAT3-dependent c-MYC transcription and consequently to impair cell viability. Altogether, these findings open a novel scenario for targeting c-MYC transcription, and it would be of the highest importance to check whether such an AMBRA1 dependence could be detected in other different c-MYC-driven tumours, such as leukaemia or gliomas [It is important to consider that c-MYC-MIZ-1 binding is critical for the development of MBubgroups . Interes gliomas , 80. Int gliomas . Loss of gliomas . Interes gliomas , 96.Moreover, considerable evidence supports the crucial roles of STAT3 in human diseases, particularly in immune and inflammatory disorders, infections and cancer . IncreasGroup3 or MBGroup4 die almost exclusively as a result of metastatic disease, which suggests that a treatment-resistant clone in the metastatic compartment results in relapse [Group3 [. Improved knowledge of some pathways  in MB has provided novel emerging therapeutic targets for circumventing chemoresistance and for restoring chemotherapeutic efficacy. For example, PARP inhibitors have shown considerable promise in the treatment of MB and inhibition of PARP has been shown to enhance TMZ activity in vitro and in vivo [Patients with high-risk MB are particularly resistant to the treatments that currently exist. A major barrier is temporal heterogeneity and patients with MB relapse . Very re [Group3 . Improve in vivo . Additio in vivo . CSCs ar in vivo . ExperimGroup3 overall survival and AMBRA1 expression, measurement of AMBRA1 levels could represent a novel relevant prognostic tool. Interestingly, Cavalli et al. have recently subcategorized 144 MBGroup3 into G3α (46.5%), G3β (25.7%) and G3γ (27.8%) subgroups with distinct genetic aberrations, activated pathways and clinical outcomes [Group3 and G3α, we found that high AMBRA1 is significantly associated with poor prognosis by univariate and multivariate analyses independently from c-MYC levels. Our findings indicate that AMBRA1 is an independent factor for poor prognosis in these subtypes, and it may be helpful in predicting the disease outcome and for managing MB patients. Until now, subgroup-targeted therapies are yet to be found; however, treatment choices based on specific subgroup features have certainly the potential to impact patient survival and avoid long-term side effects.On the same line, given the growing importance of patient stratification in MB towards a more personalized treatment , and conoutcomes . G3α mosSupplementary file1 (PDF 5206 KB)Below is the link to the electronic supplementary material.


Pleuroperitoneal communication (PPC) is an uncommon, but potentially life-threatening complication of peritoneal dialysis (PD). If a fistula does not close with conservative treatment, surgical repair is required. However, approximately half of these patients are forced to shift from PD to hemodialysis. Although it is important to confirm the site of the fistula to achieve a successful surgical treatment, this identification is more difficult in pediatric patients than in adults.We report two infantile cases of severe PPC associated with PD. In both cases, the age at onset was less than 2 years, and right-sided pleural effusion with dyspnea was observed. PPC was diagnosed by the change in color of the pleural fluid after the injection of a dye into the peritoneal cavity. Peritoneal scintigraphy and single-photon emission computed tomography and computed tomography (SPECT/CT) were performed, and these were effective in locating the fistula site. Endoscopic surgery (video-assisted thoracic surgery (VATS) and laparoscopic surgery) was performed. Indocyanine green (ICG), which was injected into the abdominal cavity, showed the exact site of the fistula. The fistula was successfully closed by attaching an absorbable sheet to it from the thoracic side and an autograft  to it from the abdominal side in one patient. In the other patient, the fistula site was resected and sutured, and reinforced with an absorbable sheet. In both cases, PD was resumed without any complication.We successfully treated two infants of PPC by endoscopic surgery. To identify the fistula site, the ICG navigation method was useful. Even in small infants, PPC can be treated successfully by endoscopic surgical repair if the site of the fistula is identified. Pleuroperitoneal communication (PPC) causing massive pleural effusion is an uncommon, but well-known complication among the nephrologists who treat renal insufficiency, and it is potentially life-threatening complication of peritoneal dialysis (PD) –3. AlthoIn pediatric cases with end-stage renal disease, PD is a well-established, preferred and safety method to maintain dialysis therapy , 7. TherIn this report, we present two infants with PPC, for whom surgical treatment was successfully performed by identifying the fistula site using currently available methods. To the best of our knowledge, Case 1 is the first reported case where the fistula site was identified from the abdominal side, and Case 2 is the youngest and the smallest patient with PPC in literature.A 1 year and 10-month-old girl with Denys–Drash syndrome, weighing 10 kg, was referred to our institute with subacute onset of respiratory distress due to the right-sided pleural effusion  were performed. Ten minutes after 99 m-Tc-Sn-colloid injection into the peritoneal cavity with dialysate, radioactivity was detected in the right pleural cavity. A hot spot remained in the medial area of the central tendon of the right diaphragm and mediastinal angle until the next day Fig. b.For definitive treatment, thoracoscopic surgery under separate lung ventilation was performed, with the patient placed in a left-sided semi-lateral decubitus position. A dialysate containing indigo carmine 20 mg/L) and indocyanine green (ICG) (0.5 mg/kg/L) was injected into the abdominal cavity to identify the leakage site. Though the pleural fluid was not stained blue, an infrared camera detected fluorescence signal in the pleural effusion  and ICG 0.5 mg/kg/L) was injected into the abdominal cavity, and the entire thoracic cavity was inspected carefully by thoracoscopy under separate lung ventilation. Although the pleural fluid was not stained, an infrared camera detected leakage of fluorescent fluid in the lateral edge of the central tendon of the right diaphragm, where small holes were present  was used for patch closure. It may be useful to observe the abdominal cavity when the communication site cannot be identified by thoracoscopy.We report two infantile cases of severe PPC associated with PD. Using endoscopic surgery and intraoperative visualization techniques (ICG navigation), we confirmed the critical communicating site and successfully closed the fistula. Even in very small infants, surgical treatment to PPC is effective, by identifying the fistula site using currently available methods.


BubR1 is an essential component of the spindle assembly checkpoint (SAC) during mitosis where it functions to prevent anaphase onset to ensure proper chromosome alignment and kinetochore-microtubule attachment. Loss or mutation of BubR1 results in aneuploidy that precedes various potential pathologies, including cancer and mosaic variegated aneuploidy (MVA). BubR1 is also progressively downregulated with age and has been shown to be directly involved in the aging process through suppression of cellular senescence. Post-translational modifications, including but not limited to phosphorylation, acetylation, and ubiquitination, play a critical role in the temporal and spatial regulation of BubR1 function. In this review, we discuss the currently characterized post-translational modifications to BubR1, the enzymes involved, and the biological consequences to BubR1 functionality and implications in diseases associated with BubR1. Understanding the molecular mechanisms promoting these modifications and their roles in regulating BubR1 is important for our current understanding and future studies of BubR1 in maintaining genomic integrity as well as in aging and cancer. Aging is one of the greatest risk factors for the development of a variety of diseases, including cancer . While iThe most well-described functions of BubR1 take place during chromosome migration and alignment in prometaphase . BubR1 sMitosis is a highly regulated process consisting of a myriad of cooperative mechanisms, many of which require dynamic control of key mitotic regulators to keep the cell in such a state that allows it to respond rapidly to new circumstances. For example, when all chromosomes are properly aligned and attached, the SAC must be quickly silenced to allow for complete initiation of anaphase . RepeateError correction and SAC responses are dictated by two properties of kinetochore-microtubule interactions: (1) the attachment state of kinetochores to microtubules, and (2) tension across the kinetochores between sister chromatids –22. UnatLike many proteins, BubR1 activity and abundance are controlled by PTMs such as phosphorylation, acetylation, and ubiquitination. PTMs are a crucial regulatory mechanism in fine-tuning BubR1 activity throughout the cell cycle, especially during mitosis . Here, wThe organization of the functional domains of BubR1 support its biological functions during mitosis. The overall domain structure of BubR1 is segregated spatially across the protein and highlights BubR1s dual role during mitosis , and ABBA motifs   . The staDocking of Plk1 on BubR1 involves the interaction between the Polo-Box domain (PBD) on Plk1 and a motif on BubR1 containing phosphorylated T620. PBDs are specialized domains that recognize specific motifs containing phosphoresidues, which is characterized by a Ser-pThr-Pro (STP) motif . The T62Phosphorylation at T620 is important for the kinetochore-microtubule stabilization activity of BubR1 but does not appear to influence its SAC function . Cells wThe KARD, spanning residues 665 to 682, is a highly conserved domain that is critical for proper BubR1 functioning . PTMs ocKARD phosphorylation is vital in the kinetochore-microtubule interaction dynamics that comprise error correction and chromosome migration to the metaphase plate. As microtubules extend from spindle poles to make contact with kinetochores, BubR1 is phosphorylated first at T620, then at KARD residues S670, S676, and T680 , 52, 53.The phosphorylated residues within the KARD are often discussed collectively as Plk1 targets, though S670 phosphorylation appears to be Plk1-independent and may instead be targeted by Cdk1 . ImportaLSPIIED)  Fig.  45]. Am. AmLSPIIUnlike Plk1 targets S676 and T680, S670 is phosphorylated by Cdk1 . It is pRecent structural data reveal a potentially important role for S670 in regulating the interaction between the BubR1 KARD domain and PP2A-B56. Crystal structure of PP2A-B56 in complex with a phosphorylated S670 KARD peptide reveals multiple electrostatic and H-bond interactions between p-S670 in BubR1 and R188 of PP2A-B56 , furtherIn contrast to S670, S676 is modified by docked Plk1 in response to a lack of kinetochore tension . UnattacAlso modified by Plk1, T680 is a critical component of the KARD and may function in a similar manner as S676 as tension-sensitive and unaffected by kinetochore attachment state . PhosphoPhospho-deficient mutations of each of these KARD residues alone results in a relatively minor defect in alignment capacity, with mutation of T680 being the most significant of the three . HoweverPhosphorylation of BubR1 at T608 has been identified as an important PTM, and its discovery and study have contributed to the enduring debate over whether BubR1 has a functional kinase domain. It has been suggested that p-T608 may be a result of BubR1 autophosphorylation, but there are also theories that T608 is targeted by Plk1 . MultiplPhosphorylation of BubR1 at T608 appears to also be dependent on CENP-E, a motor protein with important roles in chromosome migration and alignment. In particular, CENP-E has been shown to be crucial in the migration of polar chromosomes (those that are located in close proximity of either spindle pole rather than near the metaphase plate following nuclear envelope breakdown) and also contributes to the precision of metaphase plate alignment , 60. CelAlthough mitosis is well-established to be an all-or-nothing event, the SAC has been described as a graded response , 64, wheBubR1 and CENP-E are thought to colocalize at kinetochores prior to T608 phosphorylation , 61, 65.While it has not been clearly demonstrated that PP1 dephosphorylates T608, there is additional evidence supporting this notion. PP1 interacts with CENP-E in a manner that opposes Aurora B activity and has been shown to be involved in the “tug-of-war” or “push–pull” dynamic of chromosome congression . This AuThe observation that this self-executed PTM promotes Aurora B activity is noteworthy, as total BubR1 depletion is generally considered to result in increased Aurora B activity. This suggests that p-T608 exists, at least in part, to support Aurora B function. As BubR1 is well-known for its cofunction with PP2A-B56 to antagonize Aurora B activity, this PP1/BubR1 pathway further highlights the importance of PTMs fine-tuning BubR1 spatial and temporal function during mitosis. Multiple PTMs on the same protein having opposing functions is a strong indicator of the presence of a finely controlled biological switch. This concept introduces a different perspective from which to examine the role of multiple pathways converging on BubR1 during mitosis to control kinetochore-microtubule dynamics during chromosome congression.Although phosphorylation of BubR1 at T608 is an important regulator of BubR1 function, it remains to be clarified whether it is indeed the result of autophosphorylation or through an alternative mechanism. The functionality of the BubR1 kinase domain has been long debated in literature. While it was initially hypothesized to be a pseudokinase , recent Altogether, T608 phosphorylation seems to fill the role of polar chromosome rescue. It recruits SAC factors to lengthen mitotic arrest in their presence, whilst also promoting their ability to migrate. p-T608 thus provides a key reinforcement mechanism in maintaining genomic stability, without which small yet detrimental errors would be overlooked.Threonine residues 792 and 1008 are located within the BubR1 kinase domain, and both have been identified as mitosis-specific phosphorylation sites. The use of various mutants of BubR1 at these sites has revealed that phosphorylation at T792 and T1008 appears to be intricately involved in chromosome alignment, but not SAC function , 73. In 32P] ATP and various BubR1 mutants, it was found that phosphorylation-deficient amino acid substitutions at these sites result in decreased BubR1 autophosphorylation activity at T608 compared to wild type, while a phosphomimetic yielded markedly higher autophosphorylation levels. Together, these results suggest a potential role for Plk1 in regulating BubR1s alleged kinase activity. Phosphorylation at T792 and T1008 has been linked to chromosome alignment. Depletion of BubR1 yielded chromosome congression defects [Interestingly, these PTMs may contribute to subsequent BubR1 phosphorylation events, particularly autophosphorylation . Through defects , and res defects , these s defects .Less is known about phosphorylation of BubR1 at S1043, but certain aspects involving its timing and localization partners are beginning to be uncovered. In identifying non-Plk1 phosphorylation events on BubR1, phosphorylation at S1043 was shown to occur in conjunction with S670 .Currently, there is no data that identifies BubR1 binding partners that require or relate to phosphorylated S1043. However, given the similarities of S1043 and S670 in phosphorylation patterns, it is possible that these residues have common interacting kinases/phosphatases. Cyclin B1-Cdk1 may phosphorylate S1043 to contribute to kinetochore-microtubule interactions. Further studies of S1043 are required to confirm its primary function and elucidate its relationship to S670, the kinase domain, and the various binding partners of BubR1 in the context of kinetochore-microtubule stability.N-ε-lysine residues are termed acetyltransferases and deacetylases, respectively [+-dependent, suggesting that it is linked to the cellular metabolic state [Acetylation is a well-characterized PTM that is abundant in the mammalian proteome, the vast majority taking place on lysine residues –77. The ectively . This moectively . Sirtuinectively , FOXO3a ectively , 82, PPAectively  and p53 ectively . Interesic state .Although there are fewer reported BubR1 acetylation events compared to phosphorylation, they too play critical roles in the function of BubR1. Two significant sites of acetylation have been identified on BubR1, lysine 250 (K250) and lysine 668 (K668). Our current evidence of these sites suggests that they regulate BubR1 protein stability in opposite manners. Acetylation at K250 protects BubR1 from ubiquitination and premature degradation during mitosis , whereas+-dependent deacetylase SIRT2 can also target this site for deacetylation [The significance of acetylation on lysine 250 has been studied extensively and has been implicated both in the SAC and kinetochore-microtubule functions of BubR1 . Cells dtylation , though tylation .Cdc20 [Cdc20 to facilitate the ubiquitin-mediated degradation of various anaphase-inhibiting proteins, thereby keeping the cell arrested until all errors have been corrected and chromosome alignment has been successfully achieved. When K250 is deacetylated by HDAC2/3  [Cdc20. Ubiquitination of BubR1 appears to cause it and the other components of the MCC to dissociate from each other and from the APC/C. With the various substrate recognition domains freed, the APC/CCdc20 can go on to initiate anaphase. Mitosis is shortened in acetyl-deficient (K250R) expressing cells and prolonged in acetyl-mimetic (K250Q) expressing cells. Importantly, while expression of K250Q may promote chromosome congression and alignment, it may also prevent chromosomes from segregating, as cells with sustained K250 acetylation remain in metaphase for some time even after chromosome migration is complete and chromosomes are properly aligned [Mechanistically, acetylation of K250 serves to protect BubR1 from becoming a substrate of APC/CCdc20 . Protecty SIRT2) , 90, Bub aligned , 88, 89.R/+ in mice), which shows high levels of aneuploidy and spontaneous tumor development [R/+ mice displayed higher levels of Ndc80 phosphorylation than wild type controls, contradictory to p-T608, which relies on the BubR1-CENP-E relationship. This may be attributed to additional dysregulation of BubR1 function resulting from defective acetylation at K250.Less is known about the extent to which K250 acetylation is involved in BubR1s error correction function at kinetochores. Through the use of a heterozygous K250 acetylation-deficient mouse model  for ubiquitination on a designated target protein remain poorly understood in general, and methods of identification of specific lysine residue targets for ubiquitination are only now becoming better characterized. Therefore, further work is necessary to fully illuminate the ubiquitination and degradation processes controlling BubR1 stability, as well as if there are deubiquitinating enzymes (DUBs) that target ubiquitinated BubR1 to control its functions during mitosis and/or aging.Small Ubiquitin-like Modifier (SUMO) proteins are similar to ubiquitin, though they are not specifically used as tags for degradation. SUMOylation occurs on lysine residues  and is aAlthough SUMOylation of BubR1 has been described, its role in regulating BubR1 function has yet to be fully elucidated. K250 is currently the only known lysine residue on BubR1 to be targeted for SUMOylation . Of the As previously discussed, the residue modified by SUMOylation on BubR1 also serves as an acceptor site for acetylation during mitosis. Acetylation of BubR1 at K250 is required during early phases of mitosis and present throughout chromosome congression while the SAC is active, then reversed to allow SUMOylation at K250 to take place, which may be important in mitotic exit. Consistent with this, Acetyl-K250 and SUMO-K250 levels were inversely correlated in metaphase cells with aligned chromosomes . SUMOylaImportantly, linking the consequences of deficient and mimetic amino acid substitutions of K250 to an individual PTM is difficult, as this site is subject to multiple modifications. Thus far, we know K250 undergoes both acetylation and SUMOylation and may be subject to ubiquitination. It is important to note the use of K250R and K250Q substitutions in many of these studies of both acetylated and SUMOylated K250. K250R is both an acetyl-deficient and SUMO-deficient mutant. Thus, the phenotypes resulting from this substitution may not reflect the loss of a single PTM but rather the combined loss of multiple PTMs. In addition, the acetyl-mimetic K250Q may mimic acetylation but is consequently also SUMO-deficient. These factors make the study of K250 and other such sites a challenge and reinforce that identifying the enzymes responsible for the addition and removal of SUMO at K250 will be important in addressing the roles of these PTMs in regulating BubR1 function.www.phosphosite.org), di-methylation of BubR1 was found in mass spectrometry analysis on residue Arginine-919. However, a role for this modification has yet to be discerned, and further studies are necessary to deduce the purpose of methylation, as well as other common PTMs, on BubR1.In addition to phosphorylation, acetylation, ubiquitination, and SUMOylation, there are well over 200 other potential modifications , 40, somCrosstalk between different PTMs is a crucial mechanism in increasing the diversity of functional consequences associated with PTMs on a given protein. Transcription, cell cycle regulation, protein stability and DNA damage responses are a few processes that rely heavily on cooperativity and/or competition between PTMs. BubR1 is no exception to this idea Fig. . For exaMany of the PTMs on BubR1 described above cooperatively regulate BubR1 function Fig. . One impAs of now, much of the mechanistic basis of BubR1-mediated pathophysiology remains ambiguous. For instance, it is still poorly understood how MVA-related BubR1 mutations shape BubR1 function to promote aneuploidy. Similarly, it remains unclear how somatic mutations and PTMs modulate BubR1 to control genomic integrity as well as cellular senescence and the aging process. This is further complicated by our incomplete knowledge of the functionality of many other PTMs, including those that have yet to be discovered on BubR1, as well as the full complexity of the BubR1 domain structure having yet to be deciphered. Much progress has been made in determining how the BubR1 domain structure coordinates its interactive and PTM-receptive functionality Table , but cerFurther exploration of BubR1 with respect to PTMs, domain structure, and binding partners will shed more light on BubR1 function within the cell, and how dysfunction of these processes leads genomic instability, aging, and age-related diseases such as cancer.


Purpose: This study aims to elucidate the radiological outcome after Cortical bone trajectory (CBT) screw fixation and whether dual-threaded (DT) screws should be used in the fusion surgery. Methods: 159 patients with degenerative lumbar disorder who had undergone midline lumbar inter-body fusion surgery by CBT screw-fixation technique (2014 to 2018). Patient subgroups were based on single-threaded (ST) or DT screw, fixation length, as well as whether fixation involved to sacrum level (S1). Serial dynamic plain films were reviewed and an appearance of a halo phenomenon between screw–bone interfaces was identified as a case of screw loosening. Results: 29 patients (39.7%) in ST group and 10 patients (11.6%) in DT group demonstrated a halo phenomenon (p < 0.0001 ****). After subgrouping with fixation length, the incidence rates of a halo phenomenon in each group were 11.1%:3% (ST-1L vs. DT-1L), 37%:13.8% (ST-2L vs. DT-2L), and 84.2%:23.5% (ST-3L vs. DT-3L). Among the 85 patients with a fixation involved in S1, 26 patients (52%) with single-threaded screw (STS group) and 8 patients (22.8%) with dual-threaded screw (DTS group) demonstrated a halo appearance (p = 0.0078 **). After subgrouping the fixation level, the incidence of a halo appearance in each group was 25%:0% (STS-1L vs. DTS-1L), 40.9%:26.3% (STS-2L vs. DTS-2L), and 87.5%: 30% (STS-3L vs. DTS-3L). Conclusion: Both fixation length and whether fixation involved to S1 contribute to the incidence of screw loosening, the data supports clinical evidence that DT screws had greater fixation strength with an increased fixative stability and lower incidence of screw loosening in CBT screw fixation compared with ST screws. Level of evidence: 2. Spinal fusion is currently the standard surgical treatment for various lumbar spinal disorders. Pedicle screw fixation along the axis of the pedicle of the lumbar arch is the most common and reliable procedure in fusion surgery. With this entry point and route, the screw contacts the cancellous bone of the pedicle and vertebral body instead of the cortical bone of the pedicle. However, in the aging society, resorption of the cancellous bone trabeculae in osteoporosis patients who underwent pedicle screw fixation may present instrument complications, such as screw loosening or displacement ,2. CortiTo the best of our knowledge, no reports have examined the incidence of screw loosening in CBT with differently designed thread screws. Therefore, we retrospectively reviewed 159 patients with degenerative lumbar disorder and who underwent midline lumbar inter-body fusion (MIDLF) surgery by CBT screw-fixation technique with single-(ST) or dual-threaded (DT) screws from November 2014 to May 2018 and followed-up for more than 6 months. This study aims to elucidate the radiological outcome after CBT screw fixation and whether DT screws should be used in such surgery.Since November 2014, we began performing MIDLF with CBT fixation in our institute for degenerative spinal disorders because of the excellent fixation strength declared by Santoni et al. . To dateA total of 73 patients  who underwent CBT fixation with ST screws were classified into the ST group, and a total of 86 patients  with DT screws were classified into the DT group . NeitherUntil now, the National Health Insurance Administration of our country only offers ST screws for patients who need instrumentation. Therefore, patients must pay at their own expense or private insurance supported for using DT screws. We used both ST and DT screws in patients who underwent CBT surgery based on patients’ insurance situation and agreement of self-funding. The ST screw is a traditional cancellous screw used in conventional surgery with a constant pitch of 2 mm A. The DTInstead of a traditional mini-open MIDLF with CBT, we chose the minimally invasive MIDLF surgical procedure reported by Chen et al.  to achieBased on the research by Sandén in 2004, a radiolucent zone is a good indicator of the loosening of a pedicle screw . In the t test, and Fisher’s exact probability test. Significance was set at p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.Data-plotting and analysis were performed using GraphPad Prism. Data were represented as mean ± SD. All categorical data are presented as a percentage or number. The results among groups were compared using unpaired p = 0.0055 **), only 10 out of 86 (11.6%) patients in DT group exhibited halo sign or screw motion on the plain films  in contrast to 29 out of 73 (39.7%) patients in ST group and (p = 0.3179), 37%:13.8% , and 84.2%:23.5% , respectively (p = 0.0078 **) (p = 0.5147), 40.9%:26.3% , and 87.5%:30% , respectively  . They weectively , indicatDecompression with instrumented spinal fusion has become a common and reliable technique for treating a variety of spinal disorders. Many approaches have been advocated in the past decades, and posterior approaches are more familiar to most spine surgeons. Traditional pedicle screw fixation with lumbar interbody fusion usually requires longer incision and wide dissection of the muscular structure, which may cause damage to the posteromedial nerve branch crossing the facet joints and in turn lead to prolonged hospitalization and chronic low-back pain. CBT screw insertion is a novel fusion technique in spinal surgery that was first described by Santoni et al. in 2009; such a technique increased the uniaxial yield pullout load by 30% compared to TPS . SeveralSurgeons often use cancellous screw for TPS method. However, Dr. Santoni used cortical screws for CBT to maximize thread contact with the high-density cortical bone. In 2015, Ueno et al. reported that the specific trajectory itself has a major impact on the increased strength obtained by CBT compared with the differently designed thread screws . HoweverPrevious experimental studies by Mckinley  and ChenIn the present study, patients who underwent MIDLF with CBT fixation by using ST screws had more than three times of incidence of screw loosening than those who used DT screws (39.7% to 11.4%). Although the incidences both increased with fixation length , the difference became more significant when the fixation length was beyond level 3. Moreover, nearly all of the screws loosened at S1 level with the following incidences: STS-1L: 25% (3 in 12), DTS-1L: 0% (0 in 6), STS-2L: 40.9% (9in 22), DTS-2L: 26.3% (5 in 19), STS-3L: 87.5% 14 in 16), and DTS-3L: 30% (3 in 10). Only one patient of in the 3-level fixation with ST screw had bilateral S1 and one L3 screw loosening. The lumbosacral junction remains difficult to achieve for successful spinal fusion, and the prevention of construct failures, such as screw loosening at the S1  in 16, a. SakauraBesides to fixation length and fixation involved to scrum level contribute to the incidence of screw loosening. The data supports the evidence that the DT screws had greater fixation strength, with an increase in the fixative stability and a lower incidence of screw loosening in CBT screw fixation compared with ST screws. Therefore, we strongly suggest that surgeons use DT screws when performing CBT fixation surgery beyond three levels or at S1in order to reduce these incidences of screw loosening.