To investigate the correlation between postoperative limb/component alignments and clinical/functional outcomes following medial unicondylar knee arthroplasty (mUKA).Inclusion criteria included peer-reviewed English- or German-language publications assessing postoperative limb or implant alignment and clinical outcomes of mUKA. Methodological Index for Non-Randomized Studies (MINORS) was used to assess article quality. A total of 2767 knees from 2604 patients were evaluated. Significant correlations were observed between postoperative limb/component alignments and clinical/functional outcomes after mUKA. Inferior outcomes were associated with lower placement and excessive valgus alignment of the tibia component (> 3°). A recommended external rotation of 4°-5° was identified for the tibia component, with specific cut-off values for the femoral and tibia components. Optimal outcomes in mUKA were associated with a varus coronal limb alignment. The tibia implant component performed well within a specific alignment range. An exact external rotation value was recommended for the tibia component, while internal rotation correlated negatively with the femoral component.IV (level IV retrospective case series were included).The online version contains supplementary material available at 10.1186/s40634-023-00655-3. Unicondylar knee arthroplasty (UKA) is a well-established treatment option for osteoarthritis and osteonecrosis, specifically targeting either the lateral or the medial compartments of the knee joint . Long-teTraditionally, patients undergoing medial UKA (mUKA) have a preoperative varus phenotype and overall varus limb alignment. However, recent research challenges this notion, suggesting a more nuanced understanding of knee phenotypes and alignments; Hirschmann et al. analyzed 308 non-osteoarthritic knees and identified 43 different knee phenotypes, with functional and anatomical alignment targets observed in varying proportions . FurtherIt is well known that undercorrection may contribute to increased polyethylene wear, while overcorrection may lead to osteoarthritis in the lateral knee compartment , 29. AddThe objectives of this systematic review are twofold: to identify significant correlations between different postoperative limb/component alignments and clinical/functional outcomes in mUKA, and to examine whether specific postoperative coronal limb or coronal/axial component alignments yield superior clinical and/or functional outcomes compared to alternative alignments. It is hypothesized that significant correlations between postoperative limb/component alignment and clinical/functional outcomes exist in mUKA. However, it is presumed that no single postoperative limb/component alignment strategy can unequivocally be proven superior in terms of clinical/functional outcomes.A systematic literature search adhering to PRISMA guidelines  was condAfter removing duplicates and collecting all relevant articles, the studies were screened based on inclusion criteria using the title and abstract. Inclusion criteria encompassed English- or German-language publications in peer-reviewed journals that assessed the clinical and/or functional outcomes of medial unicondylar knee arthroplasty (mUKA) based on postoperative overall limb or component alignment. Studies unrelated to mUKA, pertaining to lateral unicondylar knee arthroplasty (lUKA), sagittal alignment, revision arthroplasty or failure rates were excluded. Only full-text articles with available numeric data  were considered.Next, the selected articles were independently reviewed for eligibility through full-text analysis by two authors. The same authors then manually screened the reference lists for additional articles meeting the inclusion criteria but not covered by the original search terms. In case of uncertainty regarding inclusion a third author was consulted. The endpoints of the included studies comprised postoperative limb and component alignments, various clinical and functional scores, and patient-reported outcome measures (PROMs) such as the Knee Society Score (KSS), Oxford Knee Score (OKS), Knee Injury and Osteoarthritis Outcome Score (KOOS), Western Ontario and McMasters Universities Arthritis Index (WOMAC), and Forgotten Joint Score (FJS).The methodological quality of the included studies was independently assessed by two authors using the Methodological Index for Non-Randomized Studies (MINORS) for non-randomized comparative and non-comparative clinical intervention studies . The maxRelevant information such as title, author, year of publication, study design, level of evidence, number of knees, follow-up period, patient demographics, clinical and functional outcome scores, and radiological outcomes were extracted from the selected publications by one author into a Microsoft Excel spreadsheet.p < 0.05 was considered statistically significant for data interpretation.Continuous variables were described using means and standard deviations or means and ranges, while categorical variables were reported as absolute and relative frequencies. A significance level of The initial literature search yielded 215 publications, of which 12 met the inclusion criteria  were inconsistent, with varying optimal intervals for KSS and OKS scores reported across studies Table . HoweverWhen examining the tibia component, higher values of external rotation were linked to lower KSS and OKS scores Table . Ng et aThe main finding of this review is the presence of significant correlations between different limb/component alignments and clinical/functional outcomes following mUKA. Specifically, the 1º-4º HKA alignment was reported as an optimal range for the coronal alignment of the knee, resulting in superior functional and clinical outcomes compared to other analyzed intervals. However, findings regarding the coronal alignment of the femoral component are somewhat heterogeneous and ambiguous, with different values presented by multiple authors as yielding the best outcomes. On the other hand, when discussing the coronal alignment of the tibia component, the analyzed studies predominantly suggest the -2.5º —5º (varus) interval as generating the best clinical and functional outcomes. Furthermore, worse outcomes have been observed for patients with a tibia component positioned lower than the intercondylar eminence and the lateral joint, and/or in excessive valgus alignment relative to the lower limb axis. In terms of the axial alignment of the implant, a clear recommended interval of external rotation for the tibia component has been identified as 4º-5º. Cut-off values for external rotation of the tibia and femoral components have also been determined, with 10º-12º for the femoral component and 8º-9º for the tibia component. Additionally, a negative correlation has been observed between internal rotation of the tibia component and the femoral component. In conclusion, the study has achieved its aims and partially confirmed the hypothesis.One interesting finding is the debatable reliability of the Knee Society Score (KSS) in calculating clinical outcomes, particularly the clinical/objective KSS. Several included studies question the deduction of KSS scores for varus or valgus limb alignments that fall outside the recommended intervals, such as 5º-10º valgus FTA for the 1989 KSS or 2º-10º valgus for the 2011 KSS , 16, 30.This systematic review is the first to analyze both limb and implant component alignments following mUKA from the perspective of clinical and functional outcomes. While Riviere et al. conducted a systematic review on limb alignment in mUKA ; their fHowever, this study has several limitations. One drawback is the lack of differentiation between fixed and mobile bearing medial UKA. The risks associated with overstuffing the medial compartment in mobile bearing UKA, as highlighted by Smith et al. , were noDespite these limitations, the reported results should provide guidance to orthopedic surgeons and improve the understanding of mUKA as a valid option for reducing knee pain and restoring functionality in patients with isolated medial osteoarthritis. However, future studies with higher levels of evidence and larger cohorts are needed. An international conversation should also be initiated regarding the criteria used in the KSS to award or deduct points for knee alignment. Additionally, given the existence of two KSS scores (1989 and 2011) in circulation, it is recommended that orthopedic surgeons reach a consensus and recommend the use of only one KSS while discontinuing the other. This will help prevent the dissemination of heterogeneous and contradictory results in the scientific community.Optimal outcomes in mUKA were associated with a varus coronal limb alignment. The tibia implant component performed well within a specific alignment range. An exact external rotation value was recommended for the tibia component, while internal rotation correlated negatively with the femoral component.Additional file 1.


As cell signaling research has advanced, it has become clearer that signal transduction has complex spatiotemporal regulation that goes beyond foundational linear transduction models. Several technologies have enabled these discoveries, including fluorescent biosensors designed to report live biochemical signaling events. As genetically encoded and live-cell compatible tools, fluorescent biosensors are well suited to address diverse cell signaling questions across different spatial scales of regulation. In this review, methods of examining spatial signaling regulation and the design of fluorescent biosensors are introduced. Then, recent biosensor developments that illuminate the importance of spatial regulation in cell signaling are highlighted at several scales, including membranes and organelles, molecular assemblies, and cell/tissue heterogeneity. In closing, perspectives on how fluorescent biosensors will continue enhancing cell signaling research are discussed. Cell signaling broadly describes how cells process and transmit information originating from the extracellular or intracellular environment. When the field of cell signaling was initially established, signal transduction was depicted as an essentially linear process: An external signal received at the cell surface is transduced intracellularly through a cascade of second messengers and/or other enzymes that activate effector proteins to drive a cellular response. This view typically assumed that signaling molecules are homogeneously distributed inside cells and that each distinct signal is transduced along a dedicated pathway. These early studies were instrumental in building our knowledge and laying the foundation for our understanding of signaling pathways. However, empirical observations have since demonstrated this initial, simplified model is insufficient to comprehensively describe how signaling pathways achieve specific control of cellular functions, prompting new models of spatially regulated signaling networks . AccordiThe spatial regulation of cell signaling is exerted at multiple levels. At a subcellular level, signaling pathway activity can vary greatly depending on whether pathway components are located within or on the surface of membrane-bound compartments as opposed to the cytosol. Signaling machinery can also be organized through molecular assembly to form signaling complexes with distinct activities at specific locations in the cell, helping facilitate the spatially confined production or destruction of signaling molecules by local regulatory processes, known as regulatory fencing. Finally, signaling activity can vary among cell populations and across tissues as a result of paracrine and/or endocrine effects. These mechanisms of spatial regulation ensure efficient cellular activities and facilitate acutely controlled signaling with high specificity. Loss of spatial regulation contributes to dysregulated signaling that leads to disease development . TherefoResearchers currently access numerous techniques to unravel the spatial regulation of cell signaling. Traditional biochemical techniques like cell fractionation and Western blotting remain staples of signaling research but involve lysing large numbers of cells to characterize a subset of known molecules, which limits spatiotemporal resolution and can obscure potentially important single-cell behaviors. Multiple ‘omics' technologies are also greatly contributing to our understanding of cell signaling by enabling high-throughput characterization of an array of molecules at discrete time points . ProteomAequorea victoria green fluorescent protein (GFP) into a rainbow of fluorescent protein (FP) variants transformed the study of cell signaling by enabling the development of genetically encoded fluorescent biosensors [−) [The Nobel Prize-winning discovery and engineering of osensors . The bassors [−) . A more sors [−) . Biosenssors [−) . The senAmong the simplest sensor configurations are translocation-based biosensors, in which a specific signaling protein or protein domain is fused to an FP and used to indicate the accumulation and/or distribution of a target molecule based on the redistribution of the fluorescence signal from one part of the cell to another. Many phosphoinositide biosensors feature this design, attaching FPs to different lipid-binding domains (e.g. pleckstrin homology domains) . Other tBy far the largest and most successful class of biosensors are switch-engineered biosensors. In this design, the sensing unit acts as a molecular switch and undergoes a conformational change upon detection of a biochemical event, which then modulates the fluorescence of the reporting unit. A molecular switch can be directly incorporated into an FP β-barrel, such that conformational changes will distort the FP structure to alter the chromophore environment and, therefore, FP photophysical properties  ,24. ThesSuch single-fluorophore switch-engineered biosensors are becoming increasingly popular for their high sensitivity and compatibility with multiplexed biosensor imaging . HoweverBeing genetically encodable means that fluorescent biosensors can be constructed using standard molecular biology techniques and introduced to cells as DNA, whereby the cell's own protein synthesis machinery generates the biosensor(s). Genetic encodability also offers the opportunity for enhanced spatial selectivity, as one can harness native cellular machinery to tailor biosensor localization to match the spatial scale of the biological question . For exaParallel technological advancements in molecular engineering and microscopy have enabled researchers to go beyond initial theories of linearized cell signaling to directly visualize previously undiscovered aspects of spatial regulation in cell signaling. Within the scope of this review, we highlight recent biosensor developments that reveal the spatial regulation of cell signaling at the level of membranes and organelles, molecular assemblies, or cell/tissue heterogeneity.Membrane-bound organelles represent enclosed signaling compartments whose physical boundaries sequester specific biochemical processes while excluding others. The surfaces of lipid membranes can also serve as signaling platforms with distinct properties from those of the bulk cytosolic environment. Biosensors can be directly targeted to different subcellular compartments through the inclusion of sequences encoding endogenous organelle-localization signals or by fusing a biosensor to a protein known to be enriched on or in a given organelle.Understanding the regulation of metabolic activities across organelles is a natural fit for this approach, as individual organelles engage in distinct catabolic and anabolic processes to respectively break down or build up the intracellular and extracellular environment ,40. AnabThe targeting of phosphoinositide-, Akt-, and mTORC1-specific biosensors has identified specific lysosomal regulation in PI3K signaling. Lysosomes have emerged to be a functionally important site for the Akt–mTORC1 pathway ,47. Howe3 and PIP2) are present on the lysosomal membrane to recruit Akt, using an indicator for phosphoinositides based on Akt (InPAkt). This biosensor contains a molecular switch comprising the pleckstrin homology (PH) domain of Akt linked to a negatively charged pseudoligand peptide and exhibits a C/Y FRET change upon displacement of the pseudoligand by the binding of PIP3 or PIP2 to the PH domain [Having detected Akt on the lysosomal membrane by expansion microscopy, a super-resolution method that involves sample expansion , Chen etH domain . InPAkt H domain . Indeed,H domain . OverallBeyond lysosomal PI3K signaling, studies using targeted fluorescent biosensors have also identified distinct Akt/mTORC1 regulation in the nucleus. From the earliest observations of Akt and mTORC1 in nuclear fractions of cell lysates, multiple groups have been attracted to the potential of Akt/mTORC1-mediated transcriptional regulation controlling survival, protein synthesis, and the metabolic capacities of the cell, especially considering the hyperactivation of Akt and mTORC1 in some cancers . StudiesTo approach this question, Zhou et al.  developeActing opposite mTORC1-directed anabolic signaling, adenosine monophosphate (AMP)-activated kinase (AMPK) regulates distinct catabolic processes across the cell to promote energy production . AMPK stEarly C/Y-FRET-based AMPK biosensors confirmed that metabolic stress activates AMPK in the cytoplasm and at the plasma membrane, as well as on the Golgi, ER, mitochondrial, and lysosomal surfaces ,71. One Indeed, recent work targeting an improved AMPK biosensor to various organelles has revealed distinct compartment-specific signaling kinetics, including isoform-specific and upstream-kinase-dependent effects. Using the sensing components found in previous C/Y-FRET AMPK biosensors, Schmitt et al.  were ablWhile previous investigations did not show meaningful nuclear AMPK activation in response to metabolic stress, ExRai AMPKAR facilitated robust detection of LKB1-induced nuclear AMPK activity in response to metabolic stress, as well as through direct allosteric activation. To clarify the regulation of nuclear AMPK activity, the ability of nuclear-localized or diffusible AMPKα2 to promote nuclear AMPK activity in AMPKα knockout cells was compared, demonstrating that only diffusible AMPKα2 was able to drive nuclear AMPK activity. These results helped formulate a mechanistic model of nuclear AMPK activity in which AMPK is first activated in the cytosol and then translocates into the nucleus to phosphorylate downstream targets. This model and associated methods of studying nuclear AMPK translocation and activity are particularly useful, as downstream AMPK targets that are exclusively nuclear, rather than fellow shuttling components, are under active investigation . ApplyinCarefully executed organelle- and membrane-targeting approaches can even upend established signaling paradigms, as is the case with a recent study of GPCR-mediated activation of extracellular regulated kinase (ERK) signaling. As GPCRs are an abundant family of signaling receptors targeted by over a third of existing small-molecule drugs, it is critical to understand the molecular mechanisms of GPCR signal transduction . As theiUsing the well-studied β2 adrenergic receptor (β2AR) as a model, Kwon et al.  sought tBy targeting these molecular tools to either endosomes or the nucleus, the authors sought to manipulate β2AR signaling in a location-specific manner. Responses from endosome- and nuclear (NLS)-targeted EKAR4 demonstrated that active, endosome-localized β2AR signaling machinery is required for ERK activity at both endosomes and the nucleus. In particular, biochemical experiments suggested that active, endosome-localized Gαs directly recruits the upstream components RAF1 and MEK1 to initiate endosomal ERK activity. These results delivered a new model of non-canonical GPCR-mediated ERK signaling where β-arrestin facilitates GPCR endocytosis and endosomally localized active Gαs recruits RAF1 and MEK1 to activate ERK signaling, which ultimately propagates to the nucleus. This model showed further promise when considered in the context of myelodysplastic syndrome, which describes a group of blood cancers typified by a long splice variant of Gαs and up-regulated ERK activity ; indeed,Molecular assemblies represent micro- or nanometer-scale signaling compartments that are organized directly at the protein level. In particular, the ability of many signaling proteins to incorporate into so-called ‘membraneless organelles' has attracted intense interest in the last decade, spurring investigations into their role in the spatiotemporal regulation of intracellular signaling. These dynamic assemblies, also referred to as biomolecular condensates, form through a process known as liquid–liquid phase separation (LLPS), which describes a phenomenon whereby groups of molecules overcome the tendency to be homogenously distributed in solution and spontaneously coalesce into membraneless, liquid-like droplets that are physically distinct from the surrounding environment .in vitro and in living cells. Notably, the authors took advantage of the spontaneous fragment complementation of split superfolder GFP (sfGFP) to examine the behavior of endogenously expressed RIα. In this system, the eleventh β-strand of sfGFP (sfGFP11) and the remaining ten β-strands (sfGFP1-10) are expressed as separate fragments that show no fluorescence alone but spontaneously assemble to reconstitute fluorescent sfGFP when co-expressed [11 facilitates its insertion into endogenous genomic loci using CRISPR/Cas9 technology, so that sfGFP1–10 can be reconstituted at an endogenously expressed protein of interest (POI) [Recently, work by Zhang et al.  revealedxpressed . The smast (POI) , in thisin vitro, but that this could be reversed by adding cAMP, indicating that cAMP regulates RIα LLPS. Indeed, they also found that treatment with cAMP-elevating stimuli rapidly up-regulated the formation of RIα condensates in cells. Finally, when the authors co-expressed FP-tagged RIα and PKA-C in cells, they observed PKA-C to co-phase separate with RIα, prompting them to examine cAMP and PKA dynamics directly within these phase-separated bodies [A tetrameric holoenzyme consisting of an R subunit dimer bound to a pair of catalytic (C) subunits, PKA becomes activated when cAMP binds the R subunits and triggers an allosteric switch that unleashes PKA-C to phosphorylate downstream targets. Notably, Zhang et al.  observedd bodies .1–10 to a POI endogenously tagged with sfGFP11, FluoSTEPs pair sfGFP1-10 as a partial FRET donor with a red-emitting FRET acceptor, such that reconstitution of sfGFP produces a functional G/R FRET biosensor only at the location of the endogenously expressed POI, eliminating issues related to POI overexpression.To investigate signaling dynamics associated with molecular-scale assemblies, it is important to engineer biosensors that can probe signaling activities at a comparable spatial scale. For example, analogous to how biosensors can be targeted to organelle membranes via fusion to localization motifs, biosensors are often fused to POIs to visualize nearby signaling. Resolving signaling activities at specific molecules, however, raises concerns about the biological limitations of overexpression systems . In partFluoSTEPs closely mirror the designs of existing FRET-based biosensors and can be readily generalized to detect various signaling targets . SpecificAMP signaling is generally initiated at the plasma membrane following ligand binding to Gαs-coupled GPCRs, which activate adenylate cyclases (ACs) to produce cAMP from ATP. cAMP then binds various downstream effectors, including PKA, which regulates cell proliferation, differentiation, and multiple tissue-specific processes . With huHowever, this model of PDE-driven compartmentation is contradicted by computational studies based on early observations that cAMP diffusion is unrestricted in cells . FluoresIntriguingly, the cAMP- and PKA-enriching behavior of RIα biomolecular condensates suggested that they may in fact function as a cAMP buffering system. To investigate the possible interplay between PDEs and RIα condensates, Zhang et al.  modifiedThe cAMP-nanoruler approach pioneered by Bock et al. has also recently proven instrumental in deciphering another key question in cAMP signaling, namely, how cells are able to distinguish cAMP elevations induced by specific GPCRs. To investigate this question, Anton et al.  fused EpIn addition to cAMP compartmentation, the specificity of PKA signaling is also increased through association with A-kinase anchoring proteins (AKAPs), a group of scaffold proteins that recruit PKA into discrete molecular complexes containing numerous other signaling molecules which regulate distinct functions at varying subcellular locations ,111. OveFortunately, there are now numerous super-resolution microscopy methods that push fluorescence imaging beyond the diffraction limit. For example, photochromic stochastic optical fluctuation imaging (pcSOFI) utilizes reversibly photoswitchable FPs, whose spontaneous flickering emission behavior is used to generate images with a resolution below the diffraction limit . HoweverWith the limited options for FPs that can be utilized for super-resolution applications and the Dronpa chromophore being non-critical for FLINC, Lin et al.  subsequeWhile the previous sections focused on subcellular spatial regulation of signaling activity, we now venture into cell and tissue heterogeneity, which encapsulates variation in signaling across gap junctions as well as through paracrine and endocrine effects. Investigating this scale of signaling is critical for understanding how multi-cellular organisms function as different populations of cells execute tasks and signaling activities they are phenotypically specialized to perform. This is where signaling heterogeneity arises, as a specific group of cells may initiate or direct signaling among a larger population or within a specific physicochemical environment. Fluorescent biosensors have utility at this scale to extract information about heterogeneity in cell signaling across time, whereas alternative methods can obscure cell–cell variability or lose temporal information. If observation is only possible at a few discrete time points, the selected frequency and length of observations can hinder the observation of complex temporal behaviors.in vivo cell cycle investigations by using red-shifted FPs [Cell-to-cell heterogeneity observed in various processes may be tied to cell cycle stage variation, which affects a variety of signaling activities that cells can execute . The impfted FPs .in vitro and in vivo investigations to probe connections between cell cycle status and neural and cardiac development in embryogenesis [Since the first FUCCI was reported, these fluorescent biosensors have broadly impacted both fundamental biology and translational research, particularly in stem cell and cancer research. In studies examining the effect of cell cycle stage on cell fate during embryonic stem cell differentiation, FUCCI has been used to reveal that shorter G1 length is maintained in pluripotency, with cells differentiated in early G1 showing an endoderm fate and cells differentiated in late G1 having a neuroectoderm fate . This woogenesis . Applyinogenesis ,138. In ogenesis  found thogenesis . Servingogenesis . The impFUCCI has also proven essential for directly providing new insights into mechanisms of cell cycle regulation. Proliferating cells deprived of mitogenic stimuli are typically observed to form two cell populations: one group of cells that stops dividing and another that continues through the cell cycle . The latTo investigate this observed heterogeneity in the apparent timing of the restriction point, Cornwell et al.  recentlyIn connection with cell cycle heterogeneity, it is also unreasonable to assume metabolic synchronization among a cell population similarly facing chemical and spatial limitations on growth and survival. While there are long-standing standardized benchmarks for organism-level metabolic homeostasis, describing the underlying contributions of specific tissues or even individual cells is critical for understanding metabolic disorders and developing effective drugs ,156. WitWhile our understanding of tissue-specific metabolic functions grows with AMPK and other pathways, less is known about single-cell metabolic variations. Towards this goal, fluorescent biosensors can be used to directly probe metabolites and signal transduction elements in real time to quantify metabolic heterogeneity. In a recent study from Hung et al.  energetiFurther work using common cell lines expressing AMPKAR2 treated with various oxidative phosphorylation (OP) inhibitors over several hours identified a population of OP-independent cells with little evidence of energy stress and a population of OP-dependent cells with strong AMPK fluctuations . Using aWhile we previously discussed GPCR-mediated ERK activity, ERK is primarily activated in response to growth factor signaling, an active area of interest in cancer research as tumors hijack growth factor signaling pathways to fuel their rapid growth ,163. Iniin vivo investigation of ERK activity by Hiratsuka et al. [Fortunately, fluorescent biosensors can meet the temporal need to probe ERK cell–cell signaling heterogeneity across the entire timeframe of interest to investigate when and how cells have variable growth factor-stimulated ERK activity. Based on imaging of a C/Y-FRET based ERK activity reporter with an EV linker (EKARev) over several hours, the signaling heterogeneity was first described as ERK pulses that were frequency modulated by growth factor receptor activity and amplitude modulated by the upstream kinase MEK ,166,167.a et al.  this heta et al. . In the As several groups have continued working in this area, more factors that contribute to growth factor-based ERK signaling heterogeneity have been identified, including cell density, growth factor specificity, and further downstream kinases . While sRecent investigations of ERK heterogeneity have been focused on understanding the integration of biochemical and mechanical cues in collective cell migration, critical for understanding embryonic development, cancer cell invasion, and wound healing ,175,176.Further work by Hino et al.  describeOn a technical level, fluorescent biosensors represent highly adaptable tools to quantify signaling at different scales of spatial regulation, from the tissue-level all the way down to the nanoscale . When puin vivo performance of most traditional fluorescent biosensors, owing to the scattering and absorption of visible light by complex tissues, drives the ongoing need to adapt existing fluorescent biosensor designs to use red-shifted (e.g. far-red or near-infrared) fluorophores, as well as to increase compatibility with multiphoton microscopy methods, especially in neuroscience, for in vivoimaging. The modularity and utility of biosensors facilitates their integration with numerous technologies that are being used to improve our understanding of cell signaling in the 21st century. At the molecular scale, for example, genetically encoded fluorescent biosensors have been integrated with super-resolution microscopy techniques to illuminate nanoscale signaling events [The use of biosensors continues expanding from (1) limited signaling pathways to broader signaling pathways, (2) limited localizations to a broad spectrum of protein- and organelle-specific targeting, and (3) fewer cell/tissue types to broader adoption in a wide array of primary cells and animal tissues. For animal studies in particular, the poor g events ,121. Howg events ,182–187.g events .On a biological level, fluorescent biosensor studies continue to reveal novel insights that broadly impact pharmaceutical and medical sciences. In this review, we highlighted recent applications of biosensors to describe aspects of spatial signaling regulation that are critical in numerous areas, including metabolism, cancer, and cell development. Just as the transcriptional, translational, and post-translational regulation of signaling elements are essential to study, understanding the spatial regulation of these components provides vital information to understand fundamental biological functions. Moreover, after biosensors identify signaling mechanisms in homeostatic contexts, they can be used to directly investigate contexts of disease-related deleterious signaling and to explore the mechanism of therapeutic interventions. Fluorescent biosensors thus enable us to go beyond simplified linear models of cell signaling to describe the intricate spatiotemporal regulation of signaling networks across scales that will illuminate 21st century biomedical research.


A HIV-infected female treated with a combination of emtricitabine/elvitegravir/tenofovir since 2017 presented an acute renal failure during her hospitalization for a SARS-CoV2 pneumonia. A computed tomography demonstrated left ureterohydronephrosis and ureteral stone. Fragments extracted by ureteroscopy showed a calculus composed of atazanavir and calcium oxalate. The patient's medical history showed atazanavir intake during ten years and then discontinued in 2017. This case report emphasizes that drug-induced urolithiasis should be considered when renal function declines, even far from discontinuation of atazanavir and without clinical signs of renal colitis. Moreover, identification of risk factors should alert to the possibility of drug-induced nephrolithiasis. Drug-induced urolithiasis represents 1% of all kidney stones. Protease inhibitors (PIs) are widely used in combination with other antiretroviral molecules for the treatment of human immunodeficiency virus (HIV). PI drugs are responsible for uncommon side effects. One concern associated with PIs, and especially atazanavir, is the appearance of crystalline precipitation in urine or renal tissue, leading to urolithiasis and renal dysfunction . In thisμmol/L (2.3 mg/dL) led investigation of the urinary tract. In particular, an abdominal computed tomography (CT) demonstrated a 5 mm renal calculus with ureteral dilatation and renal hypotrophy involving a chronic obstruction. The radiodensity was low (<500 Hounsfield units) suggesting the presence of a uric acid stone , with a low proportion of monohydrated calcium oxalate C1) and proteins (<30%) Figures . The spe and protThe analysis of the stone alone makes the diagnosis of drug-induced urolithiasis uncertain. The patient's medical history and the clinical context should be carefully considered to assess the degree of causality of the molecule. For example, other antiretroviral molecules (indinavir or other protease inhibitors) and antiviral molecules (acyclovir and foscarnet) but also antibacterial molecules  are known to crystallize in urine under specific conditions and initiate drug stone formation. Moreover, few other mechanisms related to the intake of certain medications  can induce kidney stones with a metabolic appearance, mainly constituted of oxalate/calcium phosphate and radiopaque. In this case, their prevalence is certainly underestimated .As illustrated in the present report, the ureteral stone was discovered four years after the ATZ discontinuation administered during eight years. The CT scan suggested that the urinary tract obstruction was chronic and that the episode of acute renal failure was more functional. Although it is difficult to assess the degree of causality of the drug, a drug-induced urolithiasis should be considered when renal function declines even far from discontinuation of atazanavir.In the last decade, a few case reports and small retrospective studies reported on renal stones related to atazanavir drug intake. HIV treatment changes rapidly, and a history of anti-HIV medication is essential to establish a causal link between taking medication and kidney stones. The causality is all the more difficult to establish notably when nephrolithiasis is revealed after several years of drug intake.The reason why atazanavir treatment is associated with kidney stones is still unknown, but previous works described that urolithiasis recurrence, a ritonavir-boosted atazanavir (ATV/r) long exposure, or high levels of bilirubinemia are all related factors of atazanavir urolithiasis , 5. Anot


Introduction: The ability to accurately predict breed composition using genomic information has many potential uses including increasing the accuracy of genetic evaluations, optimising mating plans and as a parameter for genotype quality control. The objective of the present study was to use a database of genotyped purebred and crossbred cattle to compare breed composition predictions using a freely available software, Admixture, with those from a single nucleotide polymorphism Best Linear Unbiased Prediction (SNP-BLUP) approach; a supplementary objective was to determine the accuracy and general robustness of low-density genotype panels for predicting breed composition.Methods: All animals had genotype information on 49,213 autosomal single nucleotide polymorphism (SNPs). Thirteen breeds were included in the analysis and 500 purebred animals per breed were used to establish the breed training populations. Accuracy of breed composition prediction was determined using a separate validation population of 3,146 verified purebred and 4,330 two and three-way crossbred cattle.Results: When all 49,213 autosomal SNPs were used for breed prediction, a minimal absolute mean difference of 0.04 between Admixture vs. SNP-BLUP breed predictions was evident. For crossbreds, the average absolute difference in breed prediction estimates generated using SNP-BLUP and Admixture was 0.068 with a root mean square error of 0.08. Breed predictions from low-density SNP panels were generated using both SNP-BLUP and Admixture and compared to breed prediction estimates using all 49,213 SNPs (representing the gold standard). Breed composition estimates of crossbreds required more SNPs than predicting the breed composition of purebreds. SNP-BLUP required ≥3,000 SNPs to predict crossbred breed composition, but only 2,000 SNPs were required to predict purebred breed status. The absolute mean (standard deviation) difference across all panels <2,000 SNPs was 0.091 (0.054) and 0.315 (0.316) when predicting the breed composition of all animals using Admixture and SNP-BLUP, respectively compared to the gold standard prediction.Discussion: Nevertheless, a negligible absolute mean (standard deviation) difference of 0.009 (0.123) in breed prediction existed between SNP-BLUP and Admixture once ≥3,000 SNPs were considered, indicating that the prediction of breed composition could be readily integrated into SNP-BLUP pipelines used for genomic evaluations thereby avoiding the necessity for a stand-alone software. While genomic information in livestock breeding and management has predominately been used for parentage verification and discovery as well as genomic evaluations, it also has other potential applications such as the prediction of breed composition . In the The ability to accurately predict the breed composition of an animal using genomic information has many potential uses. Firstly, genomic information can be used to verify that an animal is a purebred, thus preserving the integrity of the herd book. This may be of particular benefit for rare breeds where limited purebred breeding individuals exist or where no pedigree information is recorded and, therefore, it can help correct pedigree errors and estimate kinships when ancestry data are missing. Secondly, prediction of breed composition could assist in delivering consumer confidence in the authenticity of products from certain breeds which may command a higher market price . Furtherst and PCA have been used to select informative SNPs to discriminate between cattle breeds  which was developed to primarily increase the accuracy of genomic predictions whilest generating genotype information for mutations of interest . All aniormation .Expected breed composition was available on all animals based on their recorded ancestry; 98,883 genotyped animals from 13 breeds were expected (based on ancestry) to be purebred. Breeds included were Angus, Aubrac, Blonde d'Aquitaine, Belgian Blue, Charolais, Friesian, Hereford, Holstein, Limousin, Parthenaise, Saler, Shorthorn, and Simmental. Using the available genotypes, a principal component analysis (PCA) based on a genomic relationship matrix was calculated using the approaches described by K) from 11 to 14. K = 13 was the chosen number of breed clusters as it had the lowest cross-validation error; each of the 13 breeds separated into a distinct cluster. Individuals with a subsequent ancestry assignment of Admixture V1.3  was alsoWithin breed identity-by-state (IBS) clustering was performed on all purebred animals in Plink V1.9 , which iTo validate whether breed composition could be predicted using SNP data, a population of purebred and crossbred animals which had no direct relationship  to the purebred training population was generated. Where possible, 250 purebred-verified animals from each of the 13 breeds were included in the validation population.K = 13) was completed on all genotyped animals. The 500 purebred animals within each of the 13 breeds from the training population were fixed as purebred in a supervised Admixture analysis and the breed composition of all remaining admixed animals was predicted. Animals compromised of, at most 4 breeds were subsequently selected where each of the breeds represented had to belong to one of the 13 purebred populations included in the present study. In the two-way crosses, animals which had an Admixture breed composition prediction between 45%–55%:45%–55%, 20%–30%:70%–80% or 70%–80%:20%–30% were retained as a two-way validation population, consisting of 2,281 animals. Animals with an admixed breed composition comprised of In order to identify a known admixed population for validating SNP-BLUP and Admixture breed composition predictions, a supervised Admixture analysis  analysis as:The pairwise g breeds . The pais breeds . A phylosoftware  to visuaIISNP-BLUP using MIX99 software  was conducted in Admixture  were generated using seven different SNP selection strategies. The SNP selection population  was usedThe number of predefined SNP required per chromosome was randomly selected until each of the respective panel densities was obtained.“cluster” (V2.1.2 The partitioning-around-medoids (PAM) algorithm clusters SNPs on each chromosome together based on their proximity in genomic position, not taking LD into account. The algorithm was run for each chromosome separately with the number of clusters created per chromosome set to the number of predefined SNPs for that chromosome. The SNP located in the middle of each cluster was selected, as described by ” V2.1.2 .a) b) th highest ranking for the c) Highest ranking SNPs based on The fixation index (a) PCA ranking and block method: The SNP with the highest SNP weighting within each block was chosen.b) PCA ranking and PAM method: The SNP with the highest SNP weighting within each PAM cluster was selected.th highest ranking based on PCA SNP weightings were chosen per chromosome, irrespective of location on the chromosome, where n was the number of predefined number of SNPs for that chromosome.c) Highest ranking SNPs based on PCA: SNPs in the nSNP weightings were calculated using the “smartpca” algorithm in Eigensoft v7.2.1  applied a) SNP-BLUP variance and block method: The SNP with the largest standard deviation of SNP effects within each block was chosen.b) SNP-BLUP variance and PAM method: The SNP with the largest standard deviation of SNP effects within each PAM cluster was selected.th highest ranking based on the standard deviation of SNP effects were chosen per chromosome, irrespective of location on the chromosome, where n was the number of predefined number of SNPs for that chromosome.d) Highest ranking SNPs based on SNP-BLUP variance: SNPs in the nSNP-BLUP was used to estimate the SNP effects within the SNP selection population of each breed individually using all 49,213 SNPs. From this, the standard deviation (SD) of the BLUP model solutions per SNP were estimated within the SNP selection population of all 13 breeds and SNPs were ranked based on the SD of the SNP effect across all 13 breeds; SNPs with a larger standard deviation were given a higher ranking. Three alternative methods of picking SNPs based on using the SNP-BLUP SD were investigated.Random Forest is a machine-learning method  that emp. The regression model was run 13 times, once for each breed. Each SNP received a weighting, and SNPs which were the most informative for distinguishing between breed classes ranked highest. The highest ranking SNPs of a predefined number per chromosome were retained.Partial least square discriminant analysis (PLSDA) is another machine learning method based on the PLS approach . In the Breed composition predictions from SNP-BLUP using all 49,213 SNPs were considered the gold standard and used for comparing the prediction performance from each of the low-density panels. Animals in the purebred validation population were considered to be accurately assigned when their estimated breed proportion of a specific breed was predicted to be ≥0.90. The difference in the main breed proportion estimates for crossbred animals predicted using all the low-density panels and the gold standard 49,213 SNPs were compared. In addition, the three SNP selection methods with the smallest mean difference in breed composition predictions from the gold standard, were also used for breed composition prediction using Admixture . The AdmThe greatest genetic differentiation was observed between the Salers and both the Simmental and Shorthorns ( 0.039) . The str 0.039) . The PCAr breeds . The clor breeds .p > 0.05) from zero, suggesting that there is no systematic difference between the methods that would lead to over or underestimation of breed composition. Both SNP-BLUP and Admixture accurately assigned The mean difference in predicted breed composition between SNP-BLUP and Admixture using all 49,213 SNPs was 0.04 across both purebred and crossbreds, which was not different , how these SNPs were selected did not impact greatly the predictions.The only discrepancies observed between SNP-BLUP and Admixture breed composition predictions for purebreds using the full SNP dataset was for Blonde d'Aquitaine, Belgian Blue, and Parthenaise. Some animals in these breeds were misassigned to another closely related breed; this phenomenon may be due to the relatively smaller sample sizes of these breeds in the validation population  as well as their close genetic resemblance to other breeds. The largest discrepancy in prediction of breed composition detected between Admixture and SNP-BLUP predictions for crossbred animals was for the two-way Holstein-Friesian crosses ; becauseIn order to assess how mislabeled individuals in the training population affect breed composition predictions using SNP-BLUP and Admixture, 250 purebred Angus animals were substituted with 250 two-way cross Angus animals in the training population; they were all labelled as purebred Angus. All 250 purebred Angus animals in the validation population were predicted by Admixture to be ≥0.90 Angus. In contrast, when predictions were based on SNP-BLUP, only six of the 250 purebred Angus animals in the validation population were predicted to be ≥0.60 Angus. Therefore, SNP-BLUP appears to be more sensitive to mislabeled individuals in the training population, while Admixture was able to accurately estimate breed composition even with 250  mislabeled individuals present.SNP-BLUP required a minimum of 2,000 and 3,000 SNPs to accurately predict purebred and crossbred breed composition, respectively; the respective values for Admixture was 500 and 1,000 SNPs, corroborating results of Many factors impact the number of SNPs required for the accurate prediction of breed composition. These factors include, but are not limited to, the breeds included in the study , given tst SNP selection method for identifying informative SNPs which can be used for the prediction of breed composition in cattle has been extensively reported previously (st and PCA highest methods both chose SNPs located in close proximity and consequently in strong LD on each autosome, particularly when panel density was st method (st highest methods, these SNP selection strategies performed better at predicting breed composition than SNPs selected using the other SNP selection methods evaluated, all of which had weaker LD among SNPs. This suggests that informative SNPs for the prediction of breeds may be in LD and cluster together in close proximity along the genome, and the benefit of increasing SNP panel density was less with the PCA and Fst highest methods in comparison to the other methods that had weaker LD among SNPs. The success of the Feviously , as has eviously . Unlike eviously , a linka000 SNPs . This wa000 SNPs  and Fst t method  of rankiAlthough machine learning algorithms have been widely applied to cattle breeding for the prediction of a wide variety of traits such as lameness , longevist-based selection focused on the standardized variance in allele frequency among populations, while PCA-based selection focused on patterns in the data, identifying SNPs that had high loadings on the first three principal components, which captured the most significant patterns in the data. On the other hand, the PAM method only considered the genomic position of the SNP. st, PCA and Random Forest methods.The little overlap in SNPs selected across SNP selection approaches is likely due to the difference between the SNP selection methods used. Out of all SNP selection methods investigated, Random Forest was the only one that considered possible correlations among SNPs. FIt should be noted that ensuring purebred animals are recorded correctly and a careful selection of the most genetically diverse animals within breed to be included in the training population is crucial. As suggested by others , when prThe present study demonstrated that genomic information can be utilised in generating accurate predictions of breed composition which could potentially be useful for increasing the accuracy of genetic evaluations by being better able to fit breed covariates in an admixed population. st highest approach performed the best in terms of predicting purebred and crossbred breed composition, but only a marginal difference was observed between the performance of SNPs selected across all SNP selection methods when There was a strong similarity in predicted breed composition per animal between the SNP-BLUP and Admixture approaches investigated when panel density was ≥3,000 SNPs. This suggests that the prediction of breed composition could be readily integrated into the SNP-BLUP pipelines used for genomic evaluations thus replacing the use of a stand-alone software. Despite approximately 50,000 SNPs existing on most routinely-used genotyping panels, only small subsets of highly informative SNPs are required to accurately predict breed composition. This study provides a blueprint for the utilisation of the readily available next-generation sequencing technologies in the prediction of breed composition, by offering possible methods for how to identify the most informative SNPs and the optimum panel density. In general, SNPs selected using the F


Graphene has been under intense scientific interest because of its remarkable optical, mechanical and electronic properties. Its honeycomb structure makes it an archetypical two-dimensional material exhibiting a photonic and phononic band gap with topologically protected states. Here, we assemble colloidal graphene, the analogue of atomic graphene using pseudo-trivalent patchy particles, allowing particle-scale insight into crystal growth and defect dynamics. We directly observe the formation and healing of common defects, like grain boundaries and vacancies using confocal microscopy. We identify a pentagonal defect motif that is kinetically favoured in the early stages of growth, and acts as seed for more extended defects in the later stages. We determine the conformational energy of the crystal from the bond saturation and bond angle distortions, and follow its evolution through the energy landscape upon defect rearrangement and healing. These direct observations reveal that the origins of the most common defects lie in the early stages of graphene assembly, where pentagons are kinetically favoured over the equilibrium hexagons of the honeycomb lattice, subsequently stabilized during further growth. Our results open the door to the assembly of complex 2D colloidal materials and investigation of their dynamical, mechanical and optical properties. Colloidal particles bonding via attractive patches mimic the bonding of atoms in atomic compounds and materials. By assembling patchy particles into the graphene lattice, the authors obtain insight into lattice defects in this important 2D material. The most prominent representative, graphene, consists of a monolayer of carbon atoms bonded in a honeycomb lattice. The strong covalent bonds within the honeycomb lattice make the material particularly strong and light, while the honeycomb structure gives rise to a photonic and phononic band gap5. On a larger length scale, micrometre-size particles assembled into colloidal graphene, the analogue of atomic graphene assembled from colloidal particles, would open the door to two-dimensional multifunctional materials with photonic and phononic band gap for applications as 2D photonic and phononic crystals. However, producing large defect-free single-crystalline graphene of both atoms and colloids remains a great challenge, crucially limiting its applications.Two-dimensional materials have attracted intense scientific interest, both from an application and a fundamental point of view, offering applications from light-weight materials to optoelectronic devices. These materials combine extraordinary mechanical, optical and electronic properties compared to bulk materials6. However, while defects are introduced unavoidably during growth or added on purpose to tune mechanical and electronic properties, a comprehensive understanding of their formation is missing: because the trivalent carbon atoms or particles can arrange into a variety of polygons and structures, a coherent lattice exists even with defects, and rearrangements can take many paths. Despite advances in direct visualization of graphene defects using electron microscopy8, defect kinetics and healing remain poorly understood, and defect-free graphene challenging to produce.Structural defects are known to be central to all of graphene’s properties, enabling among others band-gap tuning in graphene-based electronic devices11. One advantage of colloidal systems is that defect formation14 and dynamics16 can be conveniently studied directly in real time with single particle resolution using optical microscopy18, which remains challenging in atomic systems, especially under the harsh high-temperature atomic deposition used for graphene growth19. The recently-gained ability to synthesize anisotropic particles23, in particular colloidal particles with attractive patches providing specific valency and bond angles, has opened a design space for assembling more complex structures such as molecule analogues26 and covalently bonded crystals28.Although colloidal particles are several orders of magnitude larger than atoms, their phase behaviour and dynamics are governed by the same thermodynamics principles. Phase behaviour of both atoms and colloids is largely governed by thermal forces, which means we can use colloidal aggregation and crystallization as a simple model for atomic crystallization29. Experimentally realizing these structures remains challenging, as they require fine control over specifically coordinated interactions, or purposeful geometric design to block kinetically favoured nonequilibrium routes, as recently shown for the realization of colloidal diamond31. In contrast to tetrahedrally coordinated diamond, graphene relies on the trivalent coordination of particles. Patchy particles with patches at 120∘ angles can mimic these covalent bonds; yet, achieving such valency and controlling these directed bonds on the scale of kBT, the thermal energy, remains challenging, but would open up the assembly of structurally complex 2D materials, and investigation of their structural and mechanical properties.Simulations and experiment have shown that these colloidal molecules can grow into larger assemblies, yielding rich structures ranging from the kagome lattice to buckyball-like clustersHere, we assemble colloidal graphene and elucidate the kinetic pathways of crystallization and defect formation of this 2D material. We form the graphene lattice using pseudo-trivalent patchy particles adsorbed at a substrate, and directly follow the crystallization, defect formation and healing with great temporal and spatial resolution using confocal microscopy. Fine control of the patch-patch bond strength allows observation of near-equilibrium assembly in analogy to high-temperature deposition of atomic graphene. From the number of saturated bonds and the bond strain, we determine the configurational energy of the lattice and follow its evolution during lattice rearrangement and healing. Our results reveal that the most prominent defect motif of colloidal and atomic graphene, a pentagon, is kinetically favoured in the early stages of graphene growth, and acts as seed for extended defects during subsequent growth. These results hint at the importance of the early stages of assembly in generating defect-free graphene.23. The synthesis yields tetrahedrally coordinated particles with a PS bulk and fluorescently labelled TPM patches  and 3-(trimethoxysilyl)propyl methacrylate (TPM) spheres by colloidal fusionhes Fig. . The parcL = 0.25 close to the critical volume fraction cL,c = 0.2736, and solvent demixing temperature Tcx = 33.95 °C, and add 1 millimolar of MgSO4 to screen the particles’ electrostatic repulsion and enhance the lutidine adsorption of the hydrophobic patches25. The suspension is injected into a glass capillary with hydrophobically treated walls  in Fig. 43. However, CVD-grown atomic graphene does not normally show vacancy defects, even though experiments show that vacancies are generated during the early stages of the CVD process44. Unlike the substrate-adsorbed colloidal particles, carbon atoms can approach vacancies from outside the plane during CVD growth, filling the vacancies with feedstock carbon44. Hence, unlike vacancies in colloidal graphene, vacancies in atomic graphene anneal in the CVD process, and irradiation or chemical treatment is used to induce them. These generated vacancies typically reconfigure to a (slightly) lower-energy structure that contains fewer dangling bonds. For instance, a divacancy can reconfigure into two 5-membered and one 8-membered ring45. In contrast, the divacancy in Fig. 46, making reconfigurations in colloidal graphene unlikely due to high energy barrier  51, or aligning graphene islands before merging57. While crystallization and defect dynamics in atomic graphene are ultimately governed by quantum mechanics, we expect that in the high-temperature limit studied here, where the quantum-mechanical states become quasi-continuous, our colloidal system provides a good model. Yet, differences arise from the different form of the potential and the different nature of the in-plane  versus interlayer bonding . Despite these differences, the energy scales are similar: at typical high-temperature deposition (~800 K) the covalent bond energy of graphene on a metal substrate is ~1 eV58, corresponding to 14.5kBT - similar to our critical Casimir bond strength of 12–15kBT. During higher temperature annealing procedures of graphene (~2000 K) the corresponding energy (6kBT) is lower, allowing together with the higher atomic attempt frequency for faster dynamics, with rearrangements occurring on the order of seconds59 rather than hours as in our colloidal case. The assembly of colloidal graphene demonstrates the increasing control over bottom-up assembly of complex materials. The honeycomb lattice is of specific interest because it is the simplest metamaterial exhibiting a photonic and phononic bandgap61, and topologically protected states. While achieving the structural complexity of macroscopic mechanical metamaterials remains still a challenge, our results demonstrate that the necessary structural motifs can be assembled using patchy particles, opening the door to microscale mechanical metamaterials4.Trivalent colloidal particles adsorbed at a substrate form the colloidal analogue of graphene, allowing direct observation of its crystallization and defect dynamics. The fine interaction control opens near-equilibrium crystallization and annealing pathways. We find that colloidal graphene defects originate in the early stages of crystallization from pentagonal motifs that are kinetically favoured over the equilibrium hexagonal motifs, further stabilized by adjacent heptagonal motifs, together forming stable grain boundaries. These results are reminiscent of high-resolution electron microscopy observations of atomic graphene, which however are limited to fully grown graphene, and cannot access the initial stages of crystallization. Grain boundaries and extended polyvacancies reconfigure towards lower-energy states by an interplay of dangling-bond saturation and lattice distortions, ultimately increasing the number of hexagons. Strategies for synthesizing large atomic graphene crystals tend to circumvent this issue by either generating as few graphene seeds as possible4 . The particles are washed several times in the water-lutidine mixture. The resulting particle dispersion is injected into a silanized hydrophobic glass capillary  and sealed with teflon grease (Krytox GPL-205). The full silanization procedure is given in Supplementary Method Tetrapatch particles  are dispersed in a binary solvent of 25% 2,6-lutidine  and 75% milliQ water with 1 millimolar MgSOT ≊ 0.6 °C), which causes one of the particle patches to attach to the sample wall. For heating, we use a well-controlled temperature stage in combination with an objective heating element to obtain a relative temperature accuracy of about 0.01 °C with minimal temperature gradients.Particles are left to sediment to the bottom of the sample at room temperature before measurements. We tilt the sample slightly during sedimentation, so a small density gradient is present in the sample. We then heat the sample to approximately 33.35 °C  to determine the centre of each patchy particle. Because one of the patches is attached to the glass, each particle centre is approximately given by this lower patch. The three remaining patches are suitable for bonding, and form a single blob with another bonded patch if bonded. This tells us if two nearby particles are actually bonded or merely randomly meeting. For more tracking details, see Supplementary Note We image the assembled structures using confocal microscope image stacks, sometimes alternating with bright field images. The images are processed using particle tracking software , and the composition of the binary solvent, as detailed in ref. 46. By formulating the critical Casimir potential model presented in ref. 46 for patchy particles and benchmarking it onto our patchy particles as shown in ref. 63, we determine the attractive minimum of the pair potential to be 12–15kBT in the studied temperature range of ΔT = 0.25–0.1K. The full potential is given in Supplementary Note The critical Casimir potential is short ranged, its magnitude and range are set by the temperature offset ΔThe bond-bending potential is harder to estimate theoretically, but can be determined from experimental measurements by following clusters of three particles, and tracking the fluctuations of the internal angle of these particles. The resulting angle probability distribution can then be converted to a potential, as shown in Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


A 25-year-old Gambian man was admitted for persistent hyperpyrexia, worsening dyspneaand asthenia, and new-onset cardiac murmur at routine control. He had no pastmedical history.At the admission, transesophageal echocardiography (TEE) demonstrated a leftventricular ejection fraction of 60% with a Sievers type 0 anterior-posterior (A-P)bicuspid aortic valve (BAV) with severe regurgitation , a 13-mm mobile vegetation on the ventricular side of the anteriorvalve leaflet , a dilatSince blood cultures were negative throughout his hospital stay, empiric antibiotictreatment with ceftriaxone and vancomycin was started.Brain computed tomography showed ischemic lesions in the frontal lobe due to septicembolism, and coronary angiography showed no abnormalities.The patient was scheduled for urgent aortic valve neocuspidization (AVNeo) and mitralvalve annuloplasty. Since aortic valve (AV) replacement with a mechanical prosthesiswas excluded due to the patient’s inability to follow anticoagulant therapy as wellas a homograft implantation due to unavailability, we preferred AVNeo to biologicalprosthesis replacement.Operation was performed through a median sternotomy and with standard aortic/bicavalcardiopulmonary bypass. An 8×8-cm piece of pericardium was harvested, thencleaned and fixed in 0.6% glutaraldehyde solution for 10 minutes and rinsed threetimes in normal saline. Subsequently, we performed aortotomy and exposed the AV andconfirmed the echocardiographic findings: BAV had severely retracted leaflets and alarge fragile vegetation on the ventricular side of the anterior cusp. After theexcision of the two leaflets, we observed a first large periannular abscess (PA)located under the anterior commissure  and a smWe also repaired the mitral valve with posterior annuloplasty using a 32-mm band ofautologous pericardium.At the end of the surgery, intraoperative TEE showed good function of the AVNeo valveand only mild regurgitation  and goodth postoperative day with indicationto follow antibiotic therapy for other four weeks and cardio aspirin for six months.No recurrence of infection occurred after one year of follow-up, and routine controlechocardiogram demonstrated good function of the AVNeo valve and mitral valve.The patient was discharged on the 141][. Ozaki et al.4][. At the end, we were also worried about the possiblerisk of reinfection due to the large PA and the presence of a biological prosthesis’sewing cuff, which might render a reoperation particularly challenging.Therefore, we had to decide between prosthetic valve replacement or AVNeo. In thepresent case, young age of the patient and specific contraindication to chronic oralanticoagulation due to social issues were considered contraindication to AVreplacement with either a biological or a mechanical prosthesis. Furthermore, thedurability of most of biological valves in young patients is comparable to themid-term result of Ozaki follow-upAVNeo surgery appeared particularly appealing in our patient since any prostheticmaterial was avoided. In fact, autologous pericardium may be less prone toinfection, may make antibiotic treatment more effective, preserve the hemodynamic ofthe aortic root, and, hopefully, will provide an adequate durability.AVNeo expert opinions retain that the goal of the procedure is a symmetrictricuspidization. The procedure may be also considered in IE, but not in annularabscess; in the latter case, however, there is no evidence.Nevertheless, we obtained an asymmetric valve due to the more complex valve anatomyof BAV Sievers type 0 A-P with the dislocation of the coronary ostia, which rendertricuspidization more difficult.Therefore, our case report demonstrates that AVNeo with autologous pericardium inyoung patients with AV IE complicated with PA may be considered a valid alternativeto biological prosthesis due to its clinical advantages considering short-termfollow-up.


During pregnancy  and the early postpartum period , participants completed online surveys. At 12 months postpartum , participants completed online surveys and a virtual laboratory visit, which included parent-child interaction tasks. We found more pregnancy-specific pandemic worries were prospectively related to lower levels of child socioemotional development based on parent report  and observer ratings , but not to parent-reported general developmental milestones. Parental emotion regulation in the early postpartum period moderated the association between pregnancy-specific pandemic worries and child socioemotional development such that pregnancy-specific pandemic worries did not related to worse child socioemotional development among parents with high  levels of emotion regulation. Findings suggest the negative consequences of parental worry and distress during pregnancy on the early socioemotional development of children in the context of the COVID-19 pandemic. Results highlight that parental emotion regulation may represent a target for intervention to promote parental resilience and support optimized child development.The COVID-19 pandemic has been linked to increased risk for perinatal anxiety and depression among parents, as well as negative consequences for child development. Less is known about how worries arising from the pandemic during pregnancy are related to later child development, nor if resilience factors buffer negative consequences. The current study addresses this question in a prospective longitudinal design. Data was collected from a sub-study ( Beginning in early 2020, the coronavirus-19 (COVID-19) pandemic upended lives worldwide with major impacts on mortality and morbidity . The panExposure to extreme or chronic environmental stress early in life, including in utero, has lifelong consequences for child development . Prior sHowever, there remains a paucity of work that has examined whether pandemic-related stress during pregnancy is prospectively related to later child development disruptions. More broadly, studies conducted prior to the pandemic have established links between perinatal distress and poorer child outcomes including worse cognitive performance , psychiaNotably, there are likely key resilience factors that could buffer any deleterious impacts of pandemic stress on child outcomes, including emotion regulation  and 12 months postpartum . We hypothesized that more pandemic worries would be related to worse child outcomes across methods . Second, we tested whether emotion regulation in the early postpartum period served as a protective factor against the effects of pandemic worries on child development. We hypothesized that emotion regulation would buffer the impact of pandemic worries, such that at high levels of emotion regulation in the early postpartum period, there would be no association between pandemic-related worries and later child outcomes. Finally, in the context of a large literature establishing significantly greater mortality, morbidity, unemployment, and burden arising from the pandemic among Black communities in the US , we ran n = 184 parent-infant dyads, who were recruited as a sub-study of a larger longitudinal perinatal cohort . At time 2, survey data was available for 833 individuals . At time 3, the sub-study recruited 219 parent-child dyads  for a virtual laboratory visit and survey (4 participants only completed the surveys). Participants who completed the child development questionnaire measures outside the recommended window for interpreting scores  and participants with GA < 37 weeks (n = 13) were excluded from analyses. Excluded participants showed higher rates of prenatal mental health symptoms and COVID-19 worries compared to the final included sample (n = 184), likely driven by pregnancy complications related to preterm birth (see Table S2).There were 1,173 individuals who completed the survey at time 1   and four pregnancy-specific worries about COVID-19  (see f items) .The Generalized Anxiety Disorder 7  assessedEmotion regulation was assessed using a 5-item scale , which wThe Survey of Well-being of Young Children (SWYC) Milestones (12-month form) was used to assess general developmental milestones . ResponsThe Ages and Stages Questionnaire: Social-Emotional, Second Edition  to 7 (very high), with higher scores indexing a greater degree of social engagement.n = 132 based on the free play task and n = 138 for the story book task, with n = 113 participants having data available for both tasks and n = 27 participants having no video available data. The most common reasons for exclusion were child or parent face/body out of view. Participants with no available video data did not differ significantly from participants with video data from one or both tasks on key study variables, including pregnancy-specific pandemic worries, general pandemic worries, anxiety symptoms, or depression symptoms in pregnancy . Inter-rater reliability was calculated on a random 20% of videotapes stratified across coders using the intra-class correlation coefficient (ICC) with absolute agreement. ICC is considered a conservative estimate of reliability because it corrects for chance agreement and takes into consideration both rank order and absolute distance between two scores  and were combined into a single observed socioemotional development measure to reflect socioemotional behaviors over multiple contexts.Videos were available for o scores . Inter-rCovariates included child age in months, child sex, parental age, parity, and EHR race . Socioecn = 184), were included in analyses using full-information maximum likelihood estimation with robust standard errors or maximum likelihood estimation  and Black , with n = 3 (1.6%) individuals identified as Black and Latinx per EHR. At time 1, 7.6% individuals were in the first trimester, 51.6% in the second trimester, and 40.8% in the third trimester. There were low-to-moderate significant bivariate correlations  and observer ratings , but not to parent-reported general developmental milestones . Although general pandemic worries were correlated with lower parent-reported socioemotional development in bivariate models  and who were identified as Black in their EHR . Older children were reported to have achieved more general developmental milestones .In a path model with all three 12 month developmental outcomes specified as correlated dependent variables and controlling for abovementioned covariates, higher pregnancy-specific pandemic worries in pregnancy were related to lower socioemotional development in children based on both parent-report  and concurrent anxiety and depression at 12 months postpartum (Tables S6 and S7). Depression  and anxiety  assessed concurrently at 12 months were also related to lower parent-reported socioemotional development. The pattern of findings was similar when we used individual reports of income (Table S8). There were no significant interaction terms between general or pregnancy-specific pandemic worry scores and parent race in relation to any developmental outcome (Table S9); the associations between pandemic worries and child developmental outcomes did not vary on the basis of race.To account for informant method effects, including the possibility that symptoms of depression and anxiety could influence parent reports of child development or interactions with children during the virtual visits, we showed that findings linking pregnancy-specific worries to both parent-reported and observed child socioemotional development were unchanged after controlling for anxiety and depression in pregnancy  and general developmental milestones , though no association was found with observed child socioemotional development   or mean  levels of emotion regulation in the postpartum period. At high levels of emotion regulation, the link was buffered   . Probing.89) see .The current study examined how an extreme stressor experienced during pregnancy was related to children’s development at 12 months of age. Our study occurred within the context of the COVID-19 pandemic and highlights the many challenges experienced by pregnant individuals . We compMore worries during pregnancy about how the COVID-19 pandemic would affect prenatal care, childbirth experience, and aspects of caring for their newborn, was linked to significantly lower levels of both parent-reported and observed socioemotional development in children. Our results support prior findings from a similar-aged sample suggesting disruptions to normative development in children born during the pandemic  and exteNotably, scores on the measure of pregnancy-specific pandemic worries were not associated with parent-reported general developmental milestones. One explanation for why pregnancy-specific pandemic worries were only linked to child socioemotional development is that the pregnancy-specific worries we identified could have subsequently evolved to parenting-related worries after birth during the transition to parenthood or the addition of a new child to the family. Increased stress or worry around parenting could have then inadvertently stymied positive parenting practices , which have specifically been shown to influence children’s socioemotional development . AnotherImportantly, we found that higher levels of emotion regulation in the early postpartum period related to positive socioemotional development and buffered against the negative effect of pregnancy-specific pandemic worries. This finding is consistent with a growing area of research demonstrating that greater parental emotion regulation is associated with benefits for child emotional development . There aThere are a few limitations to the current study. First, we relied for the most part on parent report measures, which could have introduced shared method variance  or rendered our analyses subject to informant biases resulting from psychiatric comorbidities. We addressed these issues by including an observational measure of child socioemotional development  and by controlling for symptoms of depression and anxiety at the time of pandemic worries being reported and child outcomes being reported. Nevertheless, future studies that leverage clinical assessments and reports from alternate caregivers are needed, especially since our measures of parent-reported and observed socioemotional development were not significantly correlated. Second, although we focused on a sample with approximately equal numbers of Non-Latinx White and Black participants, our sample is not representative of the wider population and findings may not generalize to other ethnic and racial groups, including those also negatively impacted by the pandemic  on parents and children. In particular, we need rigorous testing of prevention and intervention efforts that focus on building maternal resilience through better emotion regulation (e.g., In conclusion, our findings add to a growing body of literature that has both specific and general takeaways for perinatal mental health and early childhood development. In terms of specificity, we show that the lived experience of the COVID-19 pandemic may have put children at risk for less optimal developmental outcomes, particularly in the context of a parent experiencing a high degree of worry about being pregnant and caring for a newborn during the pandemic. Further work is needed to establish whether this generation of children – on e.g., , which c


Striated muscle cells, including cardiac and skeletal muscle myocytes, utilize these nuclear mechanosignaling mechanisms to respond to changes in their intracellular and extracellular mechanical environment and mediate gene expression and cell remodeling. In this brief review, we highlight and discuss recent experimental work focused on the pathway of biomechanical stimulus propagation at the nucleus-cytoskeleton interface of striated muscles, and the mechanisms by which these pathways regulate gene regulation, muscle structure, and function. Furthermore, we discuss nuclear protein mutations that affect mechanosignaling function in human and animal models of cardiomyopathy. Furthermore, current open questions and future challenges in investigating striated muscle nuclear mechanosignaling are further discussed.Mechanosignaling describes processes by which biomechanical stimuli are transduced into cellular responses. External biophysical forces can be transmitted  Striated muscles, including cardiac muscle, skeletal muscle, or insect flight muscles, are highly organized tissues specifically designed to produce precise forces and movements for a diverse set of functions. Striated muscle cells (myocytes) comprising these tissues all share the remarkable ability to both produce and adapt to highly variable microenvironmental forces. During myocyte contraction, ATP-driven myosin motors on the thick filament of the sarcomere, the contractile unit of myocytes, cyclically interact with actin filaments to induce filament sliding and force-generating sarcomere shortening . For theBoth intracellular and extracellular forces ultimately reach the myocyte nucleus, where they can be processed into biological responses that mediate the structural or functional adaptation of the cell through a process called nuclear mechanosignaling. The nucleus of striated muscle cells, among many other cell types , is now However, this homeostasis can be disrupted by defects in the ability of the myocyte nucleus to sense the mechanical forces and/or by abnormal mechanical forces imposed onto the nucleus. Myocytes under persistent abnormal mechanical forces, such as chronically altered contractility or abnormal strains imposed during passive muscle stretch, can trigger a myriad of biological responses that often lead to maladaptive cell remodeling or hypertrophy, contractile dysfunction, and cell death . In mammvia nuclear mechanosignaling. Here, we briefly review our current understanding of mechanisms by which mechanical forces and nuclear mechanosensing influence gene regulatory programs underlying pathological remodeling and dysfunction of striated muscle cells.Elucidating the role of nuclear mechanosignaling in mechanosensitive genetic signaling pathways and their involvement in muscular diseases is currently a major research area, but there are still many unknown mechanisms about the transduction of intracellular and extracellular forces into downstream gene expression In healthy striated muscle cells, the pseudo-crystalline structure of myofilaments, sarcomeres, and myofibrils establishes a highly organized and uniquely anisotropic subcellular architecture. This subcellular organization is, in part, maintained by rapid and spatially controlled protein turnover  of largeThe correct crosstalk between cytoskeleton and nucleoskeleton is necessary for proper mechanosignaling in straited muscle cells. Muscle-specific LIM domain protein (MLP) has been shown to be a stretch sensor in cardiac muscle in both mice and human . MLP KO via emerin, lamins, and many other proteins. In particular, the interaction between lamin and desmin in cardiomyocytes is critical for normal heart function. A lack of lamin A/C expression in mice cardiomyocytes has been shown to exhibit disorganized desmin filaments and paranuclear regions where the desmin filaments are detached from the nucleus  complex, which comprise many proteins that have been associated with cardiomyopathies, demonstrating its critical role in maintaining striated muscle function [please see  for revi nucleus . Many La nucleus . In a mo nucleus . Interes nucleus , suggestThus, myocytes, like many other cell types, exhibit tight mechanical coupling from chromatin to ECM. However, the anisotropic subcellular ultrastructure of myocytes and their high degree of force production impose distinct mechanical environments onto myonuclei that may elicit unique and underexplored nuclear mechanosignaling pathways underlying muscle structure and function. In the sections that follow, we discuss specific proteins known to be involved in the maintenance of this process, and the relationship between defects in nuclear mechanosignaling, mechanosensitive gene regulation and protein synthesis, and striated muscle diseases.The nuclear envelope (NE) and nucleoskeleton provide a connection between the sarcomere, the cytoskeleton, and chromatin in myocytes, which likely facilitates important mechanosensitive gene regulatory programs. In the following subsections, we discuss specific proteins known to be involved in nuclear mechanosignaling or nucleus-related pathologies in striated muscle.Nesprins provide many of the linkages between the outer nuclear membrane and cytoskeletal proteins. Nesprins generally are composed of three domains: a highly conserved C-terminus which interacts with the SUN  protein at the outer nuclear membrane (ONM), a spectrin repeat domain, and a diverse N-terminus that binds to cytoskeleton structures . NesprinNesprin-1/2 mutations have been found to be correlated with Emery-Dreifuss muscular dystrophy (EDMD) and dilated cardiomyopathy (DCM) . One patSUN1/2 are expressed in skeletal and cardiac muscles in humans . They boSUN1/2 mutations are rarely associated with cardiomyopathy . HoweverEmerin has a long N-terminus in the nucleoplasmic domain that interacts with SUN1/2, LAP1, lamin A/C, histone deacetylase 3 (HDAC3), and barrier to autointegration factor (BAF) . HDAC3 aMutations in emerin are clinically observed to be related to EDMD, DCM, and other cardiac and skeletal muscle defects . Emerin TOR1AIP1. In humans, two isoforms of LAP1 (LAP1B and LAP1C) are expressed through alternative splicing (Lamina-associated polypeptide 1 (LAP1) is encoded by the gene splicing . Both LAsplicing . It alsosplicing . Becausesplicing . HoweverPatients with LAP1 mutation p.Arg321* showed significant decrease in expression of both LAP1 isoforms in mRNA and Protein level . This spLAP2 has six isoforms derived from alternative splicing. All of the six isoforms have an N-terminus with a LEM  domain that interacts with BAF that further binds with chromatin . The C-tin vitro functional analysis shows interaction of mutated LAP2α with the pre-lamin A is significantly compromised compared to the wild-type LAP2α. Interaction of lamin A/C and LAP2α seems to be the common mechanism for LAP2α mutations that lead to diseases in other systems as well (Patients with LAP2α p.R690C mutation have been reported with severe DCM . This mu as well .The nuclear pore complex (NPC) mediates the transport of all macromolecules between the nucleus and the sarcoplasm. NPC is composed of ∼30 different proteins with multiple copies of each nucleoporin , and theMutations on NUP153 are not directly correlated with skeletal or cardiac myopathy according to our knowledge. However, skin fibroblasts with lamin A/C mutations within the Ig-fold domain, which interact with NUP153 shows altered NUP153 localization (LMNA p.453W) . Anothervia cytoskeletal protein networks to sites of myocyte damage to accelerate sarcomere repair  also can cause changes in nuclear structure. For example, a loss of filamin C in adult mouse hearts causes nuclear remodeling and altered gene expression , suggestIn the nucleus, chromatin accessibility and its three-dimensional organization are correlated . As suchMechanical stimulation also affects nuclear translocation of transcription factors. Under mechanical stimulation, myocardin-related transcription factors (MRTFs), YAP, and extracellular signal-regulated kinase (ERK) are translocated into the nucleus in myocytes . Two mecvia BAF  remain far from understood. As such, new research on the variety of other nuclear proteins and their role in regulating nuclear structure, chromatin organization, and nucleoskeletal mechanics will construct a more complete picture of the mechanosignaling processes of healthy and diseased myocytes.in vivo environment of muscles where the cells are constantly experiencing large forces and deformations. Mechanically stimulating the cells to better mimic the innate physical environment can provide more accurate representations of the mechano-sensing protein functions. Exciting ongoing research in these directions is sure to have a great impact on our understanding of the role of the nucleus in mediating muscle function in health and disease.Building upon the rich history of muscle research in which multidisciplinary approaches are embedded, combining multiple existing methodologies and technologies to answer new and exciting questions about myonuclear mechanosignaling will be undoubtedly advantageous. For example, bioinformatics  can be applied to future studies to gather a more complete picture of effects of mechanical stimulation and protein mutations in striated muscle mechano-signaling processes. These types of studies will also inform computational investigations into mechanosensitive genetic signaling in myocyte remodeling , which w