We begin the discussion by introducing recent developments in the study of such clocks and their roles at different scales and complexities. We then use current advances to elucidate the logic and molecular architecture of temporal networks that comprise autonomous clocks, providing important clues as to how these clocks may have evolved to run independently and, sometimes at the cost of redundancy, have strongly coupled to run under the full command of the cell cycle and/or the circadian clock. Next, we review a list of important recent findings that have shed new light onto potential hallmarks of autonomous clocks, suggestive of prospective theoretical and experimental approaches to further accelerate their discovery. Finally, we discuss their roles in health and disease, as well as possible therapeutic opportunities that targeting the autonomous clocks may offer.How do cells perceive time? Do cells use temporal information to regulate the production/degradation of their enzymes, membranes, and organelles? Does controlling biological time influence cytoskeletal organization and cellular architecture in ways that confer evolutionary and physiological advantages? Potential answers to these fundamental questions of cell biology have historically revolved around the discussion of ‘master’ temporal programs, such as the principal cyclin-dependent kinase/cyclin cell division oscillator and the circadian clock. In this review, we provide an overview of the recent evidence supporting an emerging concept of ‘autonomous clocks,’ which under normal conditions can be  What is an ‘autonomous clock’? An introduction to the syntax of this emerging term is essential as various fields of biological timing studies have taken on the words ‘autonomous’ and ‘clock’ in fundamentally different ways. For example, the word ‘clock’ has historically been used to define the time-keeping machinery that regulates the circadian rhythms. In this context, a clock has a strict meaning, in that it refers to an oscillator that is able to (1) free-run, (2) receive entrainment cues from the environment, and (3) compensate for changes in temperature . The emeautonomous clock to time the cyclic execution of a specific event  without clear molecular mechanisms, or functional rationales, as to the underlying biological oscillators  were mostly observed under stress conditions  when Cdk1 activity is inhibited by pharmaceutical means. These findings collectively implicate a Cdk1-independent mechanism that drives centriole biogenesis in non-dividing cells, while regulating their numbers, at least partially, in a Plk4-dependent way.As most cellular processes are intricately coupled to the progression of the cell cycle in dividing systems, what might be the functional significance of Plk4 oscillations independent of the cell cycle? In fact, the self-organizing nature of most mitotic spindles can waive the requirement for a functional centrosome in dividing cells . In the ormation . Importafrequency of these integral durations may determine the number of rounds when centrioles can be produced, while the period spent above the effective enzymatic threshold may determine the size of centrioles   , but to Although our piece particularly focuses on aspects of timing in de novo cellularization, similar questions on cell cycle autonomous timing can be proposed for other morphogenesis phenomena in early development. Indeed, a recent work has revealed the molecular basis of a long-suspected developmental clock that triggers apicobasal cell polarization independently of cell cycle progression in mice embryos , stronglSince the 1970s, genetic investigation of circadian clocks has, perhaps single-handedly, dominated the studies of biological time control at the systems level. Evidence that metabolic regulation can be periodic and autonomously regulated  i.e., t, metabolMost top-down studies in the circadian field, such as systems-level analyses concerning diurnal transcriptomes or proteomes, had historically assumed rhythms with peaks every 24 hr and largely disregarded potentially autonomous, non-circadian  rhythms. Pioneering efforts in generating robust statistical methods to study non-circadian periods with low false-discovery rates enabled the discovery of 8 and 12 hr transcriptional rhythms in mice hepatocytes . Productcan be an oscillation (detected from population-level averages), while ignoring the possibility of non-perfect sinusoids and their unique waveforms in single cells  must take the form of hierarchy with unifying properties that are independent of their specific content . For example, although DNA replication and spindle formation are processes that can operate freely in parallel  on intrinsically disordered chunks of peptides . We specIs the activity of phase-locking molecules exclusive to certain substrates in a specific temporal network, or could they be used more widely in different networks of time control? Even though potential answers to these questions can be confounded by a variety of factors related to differentiating levels of enzyme activity, or of substrate specificity, existing evidence suggests the intriguing possibility that such molecules may not be fully dedicated to their original context and might be more ‘universal’ . For exap) that is completed for a biological cycle implies a single complete period (q) for the other. There are, however, other ratios (p:q) of coupling that arise in biology. A unique example is the 1:2 phase-locking between the maturation of daughter centrioles (to become full-fledged centrosomes) and the cell cycle  and the locking strength, various phases of coupling can emerge for different p:q values. This is because increasing the locking strength often increases the span of coupling for most plausible p:q ratios . This positive relationship between the locking strength and ratios, famously called the ‘Arnold Tongue,’ is a useful graphical tool to assess the conditions under which phase-locking can emerge. Although an extended discussion of Arnold Tongues is beyond the scope of our piece, several other cases relating to phase-locking  can be qualitatively inferred from Arnold Tongues to explore underlying biological mechanisms. For further details, we refer our reader to a recent piece . For instance, the bifunctional kinase/phosphatase nature of KaiC ATPase in the cyanobacterial circadian clock is the only necessary and sufficient feature that enables the Kai ABC oscillator to run autonomously  does not prevent further lysosome biogenesis (These advances indicate that other such autoregulatory enzymes  endowed. PKC actogenesis . Whethershaded orange slice). Although this study finds a strong correlation between the variant phenotypes and KaiC ATP-ase activity to explain the vast period differences, what still remains inconclusive is whether this residue somehow directly influences the ATP-ase activity via an allosteric mechanism at the interface of two KaiC hexamers or indirectly by disrupting the formation of KaiC dodecamers, hence perturbing the clock synchronization.How could autonomous clocks set their pace? Artificially induced, or naturally occurring, gene variants that result in a dramatically long or short oscillation period may offer useful molecular insights . In termshaded purple slice). For example, the period of p53 oscillations – a response to DNA damage conserved across mammals – can vary dramatically between different species due to differing rates of p53 degradation  of these genes can continue to express periodically even in the absence of a robust cell cycle progression . This stger them . This dedirectly by the cell cycle or the circadian clock remains unknown. Except, an earlier study has postulated such an overarching, ‘energy-conservation’ function by drawing parallels between the autonomous YMCs and the circadian expressing transcriptomes from flies to humans  transcripts without any cyclic environmental or metabolic cues . If osciPlasmodium infection, its symptoms tend to occur in a cyclic manner , it is likely run by an autonomous clock that regulates their transcriptional cycles – periodic gene expression events that operate with features that resemble, but nevertheless appear to function independently of, the cell cycle and/or an innate 24 hr circadian clock , but when/if the CCO is inactivated or perturbed, they may operate independently.What would be the function of potential autonomous clocks? Are they used as fail-safe mechanisms during states of quiescence in proliferating cells ? Could they be used as pacemakers in non-dividing cells to define cell fate or terminal identity ?Do autonomous clocks act locally in subcellular settings? Could they be synchronized across the cell with intrinsic factors other than the CCO-provided cues ? Could they be synchronized across a tissue with cell-to-cell interaction mechanisms ?How could an autonomous clock evolve?Did different cellular events evolve to control time with their autonomous clocks and become progressively coupled to a common cell cycle?Was the primitive version of eukaryotic cell cycle a network of roughly coupled autonomous clocks? Did the CCO evolve to become a master regulator for this network?Are autonomous clocks just an intermediate form of cellular timing in evolution? Or are they employed actively, even in species where the CCO or the circadian clock imposes a strong degree of phase-locking?Did autonomous clocks require extrinsic cues during their evolution ?Do autonomous clocks operate with common physical or chemical principles?Could suicidal enzyme activity be a general design principle of autonomous clocks ?Are there other molecular signatures of autonomous clocks, such as DNA sequences or peptide motifs?Apart from the segmentation clock, as well as hints that suggest potential clocks for cellularization or apicobasal cell polarity, could other hallmark developmental events be regulated by their own clocks?Do potential developmental clocks crosstalk?At the systems level, similar to the autonomous 12 hr metabolic cycles in mammalians, there appears to be a third subharmonic of the circadian rhythms (~7–8 hr oscillations in gene expression). Are 8 hr oscillations independent of the cell cycle or the circadian clock? If so, what is their function?Bathymodiolus azoricus in situ in deep sea  nor synchronized between different cells in culture (en masse, even if prepared via cell cycle synchronization or light-dark entrainment, will not be sufficient to study this cell-intrinsic clock as differential phases of rhythms in individual cells will completely obscure a meaningful average and lead to a noisy flat line (Finally, although recent bottom-up efforts have provided tremendous molecular insights into how autonomous clocks may work, their identification has been limited to the interest of individual investigators. In order to scale up the discovery of potentially other intrinsic timing mechanisms,  culture . Time-lalat line . To idenlat line . Such st


Dear Editor,Simulium. Microfilariae gain access into the host system and migrate through the skin during blood meals causing severe dermatopathological conditions leading to visual impairment. The disease is also implicated in musculoskeletal pain, reduced body mass index and decreased work productivity. Onchocerciasis is a neglected tropical disease affecting more than 15 million people worldwide. Africa accounts for about 99% of active reported cases worldwide with recent records showing that over 172 million people predispose, require treatment for Onchocerciasis.The global onchocerciasis burden is caused by the transmission of microfilariae into the skin of the human host via bites from infected female blackflies of the genius At present, there is no vaccine for the prevention of the disease and treatment is done by administering ivermectin that kills only the microfilaria, but does not kill the adult worm. The microfilaricidal action of ivermectin is achieved by consistent and repeated mass drug administration (MDA). This chemotherapeutic strategy was successful in Mali and Senegal with Columbia being verified as free of onchocercaisis by WHO in 2013. However, resistance to this drug is beginning to emerge in Africa and areas coendemic to loiasis. Recently, a study showed significant induced protective immunity in a mouse model by Ov-103, Ov-CPI-2 M and Ov-RAL-2 [Onchocerca volvulus causes infection when a third-instar larva of its life cycle is transmitted to humans via the bite of the black fly, consequently resulting into onchocerciasis [Onchocerca volvulus. The proteome of the pathogen screened was found to contain 802 proteins that are non-orthologues to human proteins which were used for further screening to isolate target proteins. TargetP predicted a total of 413 sequences to have a signal peptide. However, the cleavage sites predicted by SignalP for most protein sequences were one amino acid higher than the one predicted by TargetP  alignment output showed that it had > 60% identical to four fructose-1, 6-bisphosphate aldolase of humans  BLAST output showing alignment of query  with subject  and their alignment details.


Cardiac pacemaker cells differentiate and functionally specialize early in embryonic development through activation of critical gene regulatory networks. In general, cellular specification and differentiation require that combinations of cell type-specific transcriptional regulators activate expression of key effector genes by binding to DNA regulatory elements including enhancers and promoters. However, because genomic DNA is tightly packaged by histones that must be covalently modified in order to render DNA regulatory elements and promoters accessible for transcription, the process of development and differentiation is intimately connected to the epigenetic regulation of chromatin accessibility. Although the difficulty of obtaining sufficient quantities of pure populations of pacemaker cells has limited progress in this field, the advent of low-input genomic technologies has the potential to catalyze a rapid growth of knowledge in this important area. The goal of this review is to outline the key transcriptional networks that control pacemaker cell development, with particular attention to our emerging understanding of how chromatin accessibility is modified and regulated during pacemaker cell differentiation. In addition, we will discuss the relevance of these findings to adult sinus node function, sinus node diseases, and origins of genetic variation in heart rhythm. Lastly, we will outline the current challenges facing this field and promising directions for future investigation. Pacemaker cells comprise a small population of specialized cardiomyocytes within the sinoatrial node (SAN) that spontaneously fire to trigger each heartbeat. Although pacemaker cells are contractile and have sarcomeres, they possess striking phenotypic differences from atrial cardiomyocytes, including smaller cell size with elongated morphology, cellular protrusions, and robust electrical automaticity with weak intercellular electrotonic coupling . In the Over the past several decades, a prevailing model for the cellular basis of pacemaker cell function has emerged in which mutual entrainment of a membrane clock (driven by hyperpolarization-activated cation current) and a calcium clock  confers robust automatic firing . In paraDepending on neurohormonal state and electrolyte concentration, the nodal impulse can arise from different locations within the intercaval groove, generating an electrical wavefront that ultimately exits the SAN through species-specific pathways . AlignedRemarkably, despite the anatomical and physiological complexity in heart rate regulation, both the cellular physiology of pacemaker cells and the tissue architecture in the SAN are broadly similar among diverse mammalian species despite dramatic variation in body mass and heart rate, suggesting that the determinants of cardiac automaticity constitute a robust and deeply evolutionarily conserved morphogenetic and cellular program . In keepIt is thus clear that (1) the problem of generating a robust mechanism for heart rate regulation was solved early in vertebrate evolution through the differentiation, proliferation, and architectural assembly of a specialized subtype of cardiomyocyte at the venous pole of the heart; and (2) pacemaker cells become functionally specialized by executing a conserved gene expression program with increased transcription of genes responsible for spontaneous firing and autonomic connectivity, and decreased transcription of genes associated with force generation and rapid conduction.By connecting cellular specialization at the phenotypic level to cell type-specific gene expression programs, this unified model for the origin of vertebrate heart rhythm raises several important questions that will be the focus of the present article. First, what are the upstream regulators that allow pacemaker cells to specialize and differentiate during heart development? Second, how do these regulators turn on the SAN gene program and how is this program maintained? Third, what is the relevance of these regulatory pathways to human genetic variation and human disease? Historically, our ability to address these questions has been limited by the lack of genetic tools to mark and track pacemaker cells in model systems, as well as the difficulty of isolating sufficient quantities of purified pacemaker cells for molecular analysis. Therefore, in the present work, we will also touch on technological breakthroughs in the past decade that have allowed us to define gene expression and chromatin dynamics in pacemaker cells with unprecedented genomic resolution.cis-acting genomic DNA sequences known as enhancers that can be located far from their target genes (more on this below). These binding events, in turn, permit the formation of transcriptional complexes between TF-bound enhancers and basal promoters of target genes to activate expression. The specific complement of activated transcriptional regulators available to bind genomic DNA has been termed the “nuclear regulatory environment.” The nuclear regulatory environment in a given cell, in combination with the distribution of available enhancers, determines which genes are expressed.In general, tissue-specific gene expression is accomplished through cell-type specific transcription factors (TFs) that bind combinatorially and often synergistically to in vivo global and conditional loss of function studies have been carried out in animal models with each of these factors, leading to a general model for transcriptional control summarized in this section.Pacemaker cells express a set of transcriptional regulators that are absent in atrial cardiomyocytes: Tbx3, Tbx18, Shox2, and Isl1 . These fSeveral pacemaker cell-enriched transcription factors directly repress expression of atrial-enriched genes in the SAN. By blocking differentiation toward a working cardiomyocyte cell fate, this inhibition is hypothesized to permit differentiation along the path to pacemaker cells.Tbx3, a T-box transcription factor whose expression within the SAN is conserved across several species, is the most well-characterized factor to play this role. Tbx3 binds to the regulatory elements that control expression of working myocardial genes such as Nppa and Gja5, possibly competing with Tbx5 (an activating T-box factor) to inhibit their expression and thereby inhibit chamber differentiation within the SAN  where combinations of pacemaker cell and other TFs bind directly and regulate transcription of specific genes. In the absence of such direct binding information, the connection between TFs and their targets cannot be definitively established, and a common logic to combinatorial activation of pacemaker cell gene expression cannot be defined. Thus, identifying enhancers that exhibit pacemaker cell-specific activity would add another crucial piece to this complex puzzle. Before reviewing recent experiments that have tackled this problem, this section briefly outlines our current model for how enhancers and promoters interact with each other in the context of the nucleus, and the experimental methods that can be used to identify enhancers.In mammalian cells, chromosomes each have distinct territories within the nucleus, and within each territory, transcriptionally inactive genomic loci are sequestered at the nuclear periphery, while actively transcribed regions are positioned in the interior of the nucleus . These tIn order for DNA binding TFs to access enhancers, histone proteins undergo covalent modifications at specific sites that relax their otherwise tight association with genomic DNA, which then assumes an “open chromatin” conformation . These hBecause chromatin at transcriptionally active enhancers must be accessible to DNA binding factors, the Assay for Transposase Accessible Chromatin with Sequencing (ATAC-seq) offers a conceptually straightforward method to identify regulatory elements genome-wide. In this technique, a bacterial Tn5 transposase is used to insert oligonucleotide tags into regions of accessible chromatin (locations where histones are in the relaxed or open conformation). Sequencing these regions in a given cell type allows for high-resolution determination of regions of genome accessibility. ATAC-seq datasets can be compared among closely related cell types to identify loci of differential accessibility that might contain tissue-specific enhancers . ATAC-seSeveral factors complicate the interpretation of ATAC-seq data: First, differentially accessible genome regions are not always transcriptionally active enhancers; Second, a single enhancer can have more than one gene target; Third, because most TADs contain multiple genes, it is not always clear how to assign a putative enhancer to one or more specific gene targets within its TAD. One possible experimental approach to address these problem is to define contact frequencies among distant genomic loci through chromosome conformation capture and its variants . By covain vitro strategies to differentiate pacemaker cells from induced pluripotent stem cells may provide enough material, or, alternatively, lower input chromosome conformation techniques may be become available. Until then, connecting putative enhancers in pacemaker cells with target genes will require experimental validation using in vivo models.Unfortunately, the limiting number of pacemaker cells  presents an insurmountable barrier for chromosome conformation capture, which ordinarily requires 10–20 million cells. In the future, scalable cis-regulatory architecture in pacemaker cells , showed overrepresentation of Isl1 TF binding motifs, as well as binding motifs for Meis1, a TALE-class homeodomain factor not previously studied in the context of SAN gene expression . Similarin vivo, both Galang et al. and van Eif et al. cloned selected differentially accessible ATAC-seq peaks they identified and tested them using enhancer-reporter transgenesis in mice and in zebrafish. In Galang et al., 17 differentially accessible peaks were selected and tested using EnSERT, a locus-specific transgenesis assay, or using an hsp68LacZ reporter relying on random insertion. Of these, 4 ATAC-seq peaks directed reporter activity to the SAN primordium in mouse embryos; notably, these peaks were located within TADs that encompassed Hcn4, Rgs6, Ptgfr, and Isl1, all of which are differentially expressed in pacemaker cells. The enhancer at the Isl1 locus, in particular, exhibited remarkable specificity for pacemaker cells in the developing and postnatal heart, suggesting that it functions within the pacemaker cell lineage from a very early developmental stage onward.To test whether differentially accessible ATAC-seq peaks were sufficient to function as regulatory elements In a similar vein, van Eif et al. tested 8 human differentially accessible ATAC-seq peaks located within TADs encompassing SHOX2, ISL1, and TBX3, and identified several with expression in the heart and sinus venosus in zebrafish, of which 3 had activity in the sinus venosus region of mice (2 at the SHOX2 locus and one at the TBX3 locus). Notably, the human region syntenic to the Isl1 enhancer identified in mouse pacemaker cells by Galang et al. was also strongly differentially accessible in the human ATAC-seq data from van Eif et al. providing a convincing example of cross-species conservation of genomic regulatory architecture. Indeed, both studies found that enhancer function was conserved across species to a remarkable extent – human sequences were active in mouse and fish, and mouse sequences were active in fish – often with the expected tissue-specific expression pattern. The fact that the nuclear regulatory environment in zebrafish pacemaker cells can “read out” both human and mouse DNA to achieve tissue-specific expression indicates that the regulatory kernel involving Isl1, Shox2, and other pacemaker cell TFs is an ancient vertebrate regulatory module that has been conserved over hundreds of millions of years.Isl1-Locus SAN Enhancer, or ISE) was deleted using CRISPR-Cas9. Homozygous ISE/ΔISEΔIsl1 mice were born in Mendelian ratios and were viable and fertile with no major cardiac structural abnormality by echocardiography. However, detailed molecular and histological studies showed that Isl1 protein and mRNA expression were reduced specifically in ISE/ΔISEΔIsl1 pacemaker cells, with a corresponding hypoplasia of the SAN and reduced pacemaker cell number (ISE supports Isl1 transcription during SAN morphogenesis to ensure that the SAN achieves its normal size and cellularity, allowing for normal SAN function.To test the functional relevance of these enhancers, both Galang et al. and van Eif et al. deleted regulatory sequences from the genome of mice to test whether target gene expression and SAN development are affected. In Galang et al., a 2.7-Kb segment at the Isl1 locus (hereafter, l number . Furtherl number . Taken tShox2 locus showed loss of Shox2 expression in the SAN, with dysregulation of Shox2 target genes, hypoplasia of the SAN, and embryonic lethality presumed due to bradycardia. In a parallel experiment, a 280-kb putative regulatory region containing differentially accessible ATAC peaks was also deleted from the Tbx3 locus. Homozygous mutant mice had absent Tbx3 expression in the SAN and exhibited perinatal lethality, while heterozygous mice survived to adulthood and exhibited prolonged SAN recovery times with programmed stimulation, indicating an important role for this enhancer in regulating SAN electrophysiology.van Eif et al. deleted larger genomic regions within the SHOX2 and TBX3 TADs that contained differentially accessible ATAC-seq peaks. Examination of knockout embryos from a 250-kb deletion mouse line at the In vivo studies have also demonstrated Mef2’s upstream role in regulating both SAN and non-SAN genes, similar to Tbx5 and Gata4 (By combining data from conditional loss of function studies for critical TFs, available ChIP-seq datasets for these factors, and the insights gleaned from the recent epigenetic profiling studies, a working model for transcriptional regulation in pacemaker cells can be constructed . Based ond Gata4 . RemainiUltimately, a major goal of dissecting regulatory networks in pacemaker cells is to gain a better understanding of the pathophysiology of sinus node dysfunction and, more generally, to provide insight into the genetic underpinnings of variation in human heart rhythm. A number of previous studies have related genetic variation in heart rhythm parameters to regions of non-coding DNA that were subsequently found to contain enhancer that regulate expression of nearby genes . Indeed,TBX3 enhancers that were identified and validated in In keeping with these findings, GWAS that have been performed using resting heart rate or heart rate recovery after exercise have found numerous “hotspots” in non-coding regions of the genome that were presumed to have regulatory function . One sucp value threshold needed to establish a significant association was reduced. Thus, by focusing only on differentially accessible peaks that are open in pacemaker cells, Galang et al. worked from the presumption that ATAC-seq peaks were more likely than other regions of the genome to harbor heart rate-associated SNPs, prompting them to perform a new association study on UK Biobank participants, limiting the analysis to SNPs occurring within 500 bp on either side of the human genomic regions syntenic to mouse pacemaker cell ATAC-seq peaks. The results of this analysis demonstrated many associations between heart rate and SNPs occurring near ATAC-seq peaks that are close to established genes that regulate heart rate. Furthermore, by focusing the association study on a subset of SNPs, and hence reducing the number of hypotheses tested, the uncorrected The data summarized in this article have brought us closer than ever before to a detailed understanding of how pacemaker cells acquire their unique phenotypes during development, and to connecting the genetic determinants of pacemaker cell biology to vertebrate evolution and human physiology. Nevertheless, critical knowledge gaps remain.First, while ATAC-seq data establish the genomic locations of the regulatory elements important for pacemaker cell-specific gene expression and indicate some of the transcription factors that are likely to bind to them, further work is needed to establish the precise timing and location of transcription factor binding events onto these enhancers. For example, which enhancers are specifically bound by Isl1 and what are its binding partners? What are the pioneer factors that open chromatin to allow for execution of the pacemaker cell gene expression program and what are the signaling pathways that activate these factors during sinus node development? Are these same pathways required for maintenance of pacemaker cell identify in the adult heart? How is epigenetic and transcriptional control related to age-related SAN dysfunction? Answers to these questions will fill in the missing pieces in our model for transcriptional control in pacemaker cells and will point the way toward translational strategies that could reverse SAN failure or even regenerate pacemaker cells.A second area that will require further exploration is the connection between human genetic variation and SAN disease. As discussed, genome-wide and more limited association studies have demonstrated convincingly that a significant portion of human variation in SAN behavior is attributable to genetic differences at enhancer loci that regulate expression levels of critical SAN genes. A remaining challenge will be to take these analyses a step further by exploring whether variation in human non-coding regions is associated with the development of age-related or premature-onset sinus node dysfunction, atrial arrhythmias, and need for pacemaker implant. Defining the nature of these associations and the regulatory pathways involved could lead to new targets for interventions to prevent or treat SAN disease and other atrial arrhythmias.in vitro system to model pacemaker cells, and the limited access to human primary pacemaker cells. Fortunately, new tools and approaches are rapidly becoming available to surmount these technical hurdles. First, the development of human iPSC-based in vitro differentiation protocols, as highlighted in the study by van Eif et al., allows for the possibility of exploring the functions of cis-regulatory elements and transcriptional regulators in a more realistic human context without the limits imposed by low cell numbers. Thus, techniques such as ChIP-seq and Hi-C will eventually be used to create detailed, functionally annotated genomic maps of pacemaker cells. These datasets can then be integrated with data emerging from increasingly large cohorts of genotyped patients, including those with whole-genome sequencing alongside clinical data, to provide us with a detailed high-resolution model for how the epigenome and transcriptional biology connect with disease.Historically, research on these aspects of pacemaker cell and SAN biology has been challenging because of the difficulty of isolating pacemaker cells in large numbers, the lack of a suitable in silico examination of cellular subtypes and cellular differentiation. Used in combination with new technologies that can isolate individual nuclei from differentiated tissues, these techniques will be invaluable as individual genes and pathways are explored in loss- and gain-of-function animal models and in small samples derived from human tissue. Taken together, the combination of recent technical and scientific advances leaves the field poised for rapid progress in the next several years.Alongside these technical innovations, the development of single cell genomic profiling technologies, including scRNA-seq and scATAC-seq, will circumvent the need to purify primary pacemaker cells for molecular analysis. Furthermore, data using these techniques can now be integrated across different platforms, allowing for more powerful Over the last several decades, an integrated model for the electrophysiological basis of pacemaker cell automaticity and SAN physiology has taken shape, but a detailed understanding of the underlying gene regulatory networks has remained elusive. Recent advances in developmental biology, chromatin biology, bioinformatics, and human genetics have now revealed that several key transcriptional regulators and genomic loci are critical for pacemaker cell development, SAN formation, and SAN function, and have clarified their relevance to human variation. Work in the coming years will translate these findings to an improved understanding of SAN disease, novel targets for therapeutics, and possibly SAN regeneration.RM and VV wrote the manuscript and prepared the figures. CJ prepared the figures, performed the imaging and image processing, and edited the text for intellectual content. All authors contributed to the article and approved the submitted version.VV received research support from Amgen and a consulting fee from Merck for an unrelated project. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Seizures are common in newborn infants with hypoxic-ischemic encephalopathy and are highly associated with adverse neurodevelopmental outcomes. The impact of seizure activity on the developing brain and the most effective way to manage these seizures remain surprisingly poorly understood, particularly in the era of therapeutic hypothermia. Critically, the extent to which seizures exacerbate brain injury or merely reflect the underlying evolution of injury is unclear. Current anticonvulsants, such as phenobarbital and phenytoin have poor efficacy and preclinical studies suggest that most anticonvulsants are associated with adverse effects on the developing brain. Levetiracetam seems to have less potential neurotoxic effects than other anticonvulsants but may not be more effective. Given that therapeutic hypothermia itself has significant anticonvulsant effects, randomized controlled trials of anticonvulsants combined with therapeutic hypothermia, are required to properly determine the safety and efficacy of these drugs. Small clinical studies suggest that prophylactic phenobarbital administration may improve neurodevelopmental outcomes compared to delayed administration; however, larger high-quality studies are required to confirm this. In conclusion, there is a distinct lack of high-quality evidence for whether and to what extent neonatal seizures exacerbate brain damage after hypoxia-ischemia and how best to manage them in the era of therapeutic hypothermia. Loss of oxygen (hypoxia) and blood supply (ischemia) to the brain can occur before, during or shortly after birth. Moderate to severe hypoxic-ischemic encephalopathy (HIE) occurs in ~1 to 3/1000 live births in developed nations and can lead to death, or in survivors, brain damage with lifelong disability, including cerebral palsy and epilepsy . There aSeizures are common in newborn infants with HIE. For example, in a study of electroencephalographic (EEG) monitoring in 47 neonates with HIE, 62% had seizures ,6. HowevThe mechanisms of seizures in the neonate appear to be different in many ways to those in the adult or even during childhood. The majority of neonatal seizures occur as a transient response to acute cerebral pathology and are most commonly associated with HIE 38%), ischemic stroke (18%) and intracranial hemorrhage (11%) %, ischem.- concentration resulting from high Na-K-2Cl (NKCC1) expression, which mediates Cl- entry and low K-Cl cotransporter KCC2 expression, which mediates Cl- exit from cells . Su. Su25]. 2, as measured by laser Doppler on the parietal cortex . Dur. Dur3]. There is some evidence to suggest that early phenobarbital can augment the beneficial effects of therapeutic hypothermia. In P7 rats exposed to hypoxia-ischemia induced by single carotid artery ligation and inhalation hypoxia, phenobarbital augmented hypothermic neuroprotection . SimilarThere is increasing clinical and preclinical evidence that therapeutic hypothermia per se reduces seizure activity in addition to its long-term neuroprotective effects. A study of 107 neonates with HIE showed that although hypothermia did not reduce the number of neonates with seizures, it was associated with a significant reduction in seizure burden in neonates with moderate, but not severe, HIE  60]. Fu. Fu60]. n = 53) where neonates with HIE who have already received phenobarbital were given additional phenobarbital and bumetanide or placebo reported an additional reduction in seizure burden associated with bumetanide [By contrast, a clinical pilot study of 14 neonates with HIE who were administered the loop diuretic bumetanide, which activates both renal and cerebral NKCC1, in conjunction with therapeutic hypothermia and phenobarbital was terminated early due to lack of prespecified efficacy and the finding that 3 infants developed hearing loss likely related to oto-toxicity, leading to an unacceptable risk to benefit ratio . A more metanide . HoweverKey challenges to improving management of seizures include the poor efficacy of current anticonvulsants for controlling neonatal seizures, the lack of evidence that they improve long-term outcome, concerns over adverse effects and uncertainty over their pharmacokinetics when administered in conjunction with therapeutic hypothermia. Given these issues, designing trials of new anticonvulsants in the hypothermia era using an “add-on” method, in combination with both hypothermia and phenobarbital is less than ideal and is likely to leave many important questions unanswered. Given that hypothermia is associated with reduced overall seizure burden, improves long-term outcomes and is safe for use in neonates with HIE in intensive care, we propose that therapeutic hypothermia alone should be considered to be the standard treatment rather than hypothermia plus phenobarbital, and that anticonvulsants should be randomized on top of hypothermia . This woAnimal studies suggest that anticonvulsants have the potential to cause unwanted side-effects in the developing brain, including widespread apoptotic neurodegeneration and altered neuronal proliferation. For example, in the neonatal macaque, administration of phenobarbital followed by infusion of midazolam over 5 or 24 h was associated with widespread apoptosis affecting both neurons and oligodendrocytes, with more extensive neurodegeneration after longer exposure . FurtherAlthough it is difficult to determine whether anticonvulsants have acute neurotoxic effects in human neonates, long-term exposure to phenobarbital for the management of febrile seizures, was associated with an impairment in the development of language/verbal skills by school age with no difference in the recurrence of febrile seizures . In a seCurrent anticonvulsant protocols are clearly far from optimal for use in neonates—they show limited efficacy, have high potential for adverse effects and there is a striking lack of evidence that they improve long-term outcomes. Preclinical neurophysiological studies are essential to help to better understand the pathophysiology of seizures occurring in the neonatal brain compared to the mature brain and identify specific anticonvulsant treatment strategies for the neonatal brain. High quality translational studies will help to answer questions around whether prophylactic anticonvulsant treatment is neuroprotective and more effective than delayed administration and whether anticonvulsants are associated with neurotoxic effects when given in combination with therapeutic hypothermia. Well-designed randomized controlled trials, taking into account the use of therapeutic hypothermia, are essential to develop evidence-based treatment strategies for the neonate with seizures.


Microbiome engineering is increasingly being employed as a solution to challenges in health, agriculture, and climate. Often manipulation involves inoculation of new microbes designed to improve function into a preexisting microbial community. Despite, increased efforts in microbiome engineering inoculants frequently fail to establish and/or confer long-lasting modifications on ecosystem function. We posit that one underlying cause of these shortfalls is the failure to consider barriers to organism establishment. This is a key challenge and focus of macroecology research, specifically invasion biology and restoration ecology. We adopt a framework from invasion biology that summarizes establishment barriers in three categories: (1) propagule pressure, (2) environmental filtering, and (3) biotic interactions factors. We suggest that biotic interactions is the most neglected factor in microbiome engineering research, and we recommend a number of actions to accelerate engineering solutions. Microbiome engineering is a rapidly evolving frontier for solutions to improve human health, agricultural productivity, and climate management. Microbiome engineering seeks to improve the function of an ecosystem by manipulating the composition of microbes. Two major challenges for successful microbiome engineering are 1) the design of a microbiome with improved function and (2) the establishment of an improved microbiome in a recipient system of interest. While multiple articles and reviews have addressed functional design  the desi, microbiTwo general engineering strategies are to manipulate indigenous microbes  or to inThe problem of organism establishment in recipient ecosystems is not unique to microbiome engineering; it has roots in macrobiology, particularly invasion biology and restoration ecology. We propose that adopting a cross-disciplinary conceptual framework to identify barriers to organism establishment, and then prioritizing these barriers through targeted research will accelerate successful microbiome engineering. In addition, recognizing differences in terminology and experimental design within and across disciplines will facilitate research integration across diverse ecosystems and scales. The components of a more holistic strategy are discussed below.Pinpointing and overcoming barriers to inocula establishment are important research priorities for successful microbiome engineering. Barriers to establishment have been studied extensively in both invasion biology and restoration ecology. Invasion biology aims to understand the mechanisms that promote or deter invasive species establishment . RestoraA useful theoretical framework for species establishment in microbiome engineering would identify the abiotic and biotic levers that engineers could use for successful community manipulation. Frameworks that vary in granularity have been developed within invasion biology. At one end of the spectrum, one synthesis outlined 33 mechanistic hypotheses for establishment success . At the Propagule pressure (PP) is a measure of dispersal used to describe the magnitude (dose) and pattern of the arrival (frequency) of invasive individuals. PP is one of the most commonly tested factors in macroorganism invasion biology and is often linked to invasion success . It alsoThere are a number of mechanisms by which PP in theory could influence establishment. Increasing PP can overcome the effects of ecological drift—random births and deaths that change the relative abundances of species in a community over time—also known as demographic stochasticity, which generally increases for smaller populations such as newly introduced species , 22. IncDelivery mode may influence establishment by affecting dispersal range or dose viability. If the delivery mode is insufficient, inoculants may not reach the intended location of establishment or may be debilitated when they arrive. Parallels in plant invasion biology and restoration ecology include natural seed coats that enhance plant dispersal range by animal vectors  or artifEnvironmental filtering (EF) refers to the selection of organisms that are compatible with the existing environment or are able to rapidly modify local conditions to fit requirements. Incompatible immigrants become extinct. EF can limit invasive plant establishment  and oftA lack of niche space for an inoculant is an obvious barrier to establishment. Understanding the temporal and spatial distributions of niche availability may be key to tailoring inoculant establishment . IdentifBifidobacterium infantis strains in infant guts [Bacteriodetes strain in mice guts [Alternatively, a niche can be created. Niche availability can be manipulated in some cases by altering the abundance of a single or multiple resource(s), as is done with prebiotics . In hostant guts . Similarant guts . Prebiotice guts  and xenoice guts . Continuice guts . Conversice guts . An alteice guts  or a nicice guts , see BioDisturbance, here defined as perturbation of physical  or chemical  properties of the environment, has the potential to increase species turnover. Consequently, disturbance is either a useful tool to displace unwanted residents ; see BioThe variety of BI is complex but can be broadly categorized as either antagonistic or facilitative . Within The complexity of competition can be categorized by the mechanisms organisms use to capture growth limiting resources. In the simple case, competition involves differences in search capability  and/or resource capture efficiency . Increasing the number of competitors for a growth-limiting substrate may reduce the ability of an inoculant to capture a sufficient quota of resource to survive . A generBacillus [Streptomyces [Vibrio cholerae growth [Complex competition involves additional strategies to undermine competitors. Strategies include secretion of antibiotics, signaling compounds that adversely affect the metabolism of other species , or siderophores that create new growth limitations for competitors. Antibiotic production is the best studied strategy. In pairwise interactions of cultured isolates, antagonistic antibiotic production occurred in approximately half of Bacillus  and Streptomyces  isolatesptomyces , presumae growth . The exte growth  and mighe growth .Another type of antagonistic interaction that may influence the ability of inoculants to establish is predation. Predation by protists, bacteria, and fungi is likely to be density dependent, not taxon-specific, although cases of the latter have been documented . PredatiStable facilitative interactions between microorganisms are only expected in restricted cases . For exaThe framework of PP, EF, and BI is a simple way of categorizing a long list of ecological phenomena that can be barriers (or engineering tools) for establishment of inocula in resident communities Table . The oveDecision trees are increasingly being used in macrobial ecology to manage species invasions and ecological restoration. For example, some decision trees prioritize actions based on the potential severity of invasive species on ecosystem services . In a regeneral rules for decision trees, but such a pursuit is inefficient without planning for quantitative synthesis.Building decision trees in microbiome engineering requires studies that test the relative importance of factors on establishment success. For example, a recent study found that the relative importance of PP and BI varied by organism domain, bacteria versus fungi . AnotherOverall, we posit that increasing focus on BI will lead to increased success in inoculant establishment and improve outcomes in microbiome engineering Fig. . While BIn order to confirm the role of BI in microbiome engineering outcomes and refine a decision tree of barriers to organism establishment we recommend a number of actions. First, greater attention is needed in microbial research to measure establishment (persistence and/or proliferation) of inocula over long timescales  with whole-community measurement techniques that offer broader insight and more standardized experimental design and reporting to facilitate meta-analyses. Second, a careful cross-discipline synthesis—i.e., meta-analyses of microbial studies that measure the dependence of inocula establishment on aspects of BI, as well as PP and EF, that would illuminate knowledge gaps is needed. We expand on these concepts in the sections below.To accelerate microbiome engineering across different applications, there is a need to target knowledge gaps that are contingent on experimental design. Gaps range from complexity of the inoculum to multifactorial manipulation strategies. We highlight four gaps. (1) To date, most microbial establishment studies have one or a few inoculant species. In contrast, transplant of entire microbiomes  is an increasing practice for some medical and wastewater treatment applications, respectively, yet knowledge of how these complex invasions impact community composition and functioning is limited. (2) Often microbiome engineering studies measure functional changes, not inoculant establishment. Monitoring inoculants is needed to determine if failure to achieve desired functional changes is due to lack of establishment or instead, to attenuation of desired functions in established inoculants. (3) More insight into the temporal dynamics of functional changes is needed. It is common practice to assess function at only one or two timepoints. Furthermore, the time interval between manipulation and functional assessment varies from hours to weeks. In many studies functional measurements occur immediately following probiotic intervention and longer-term impacts are not assessed, limiting insights into barriers to establishment. (4) It is likely that multiple factors simultaneously contribute to the failure or success of species establishment . Thus, Prioritizing establishment barriers through cross-discipline synthesis depends on the capacity to find related knowledge that is obscured by discipline-specific jargon. For example, medical research on microbiome engineering typically uses the term “probiotics” , while aSome standardization of experimental design features  can also facilitate synthesis. At present, wide variation in experimental design  impedes Microbiome engineering is a rapidly expanding field. There are notable cases of success in microbiome engineering for human health, in particular in the infant gut , 65, bioTable S1Table S2


Since the outbreak of the COVID-19 pandemic, there has been heightened attention towards population mental health due to the pandemic-related stress induced by profoundly changed life circumstances with recurrent lockdown situations worldwide. Representative data assessed early after the outbreak of COVID-19 show an overall increase in mental distress in the general population, and they also support that population subgroups are affected differently . There iSuch findings on sex differences of the adverse effects of the COVID-19 pandemic are pivotal to inform policy decisions as well as prevention and health service strategies. However, it might be worthwhile to take a step back and reflect on this potential sex difference, especially as this is rarely discussed in the original research papers. Firstly, is it really the case that females are more vulnerable to experience mental health deterioration under the pandemic? And if yes, what are the underlying causes of this sex difference?Regarding the first question, it is important to consider common methods of mental health assessment: most studies focus on symptoms of anxiety and depression and do rely on screening or brief self-report instruments . While tRegarding the second question, it is important to consider potential underlying causes of women tending to report higher mental health burden in surveys. Some of them might lie outside biological sex differences and might again draw back on societal gender roles and socioeconomic differences between men and women. While there is substantial evidence indicating that women report greater fear and anxiety, this may be due to a broad range of biological influences, temperamental and cognitive factors, as well as socialization processes. A large representative survey identified people caring for pre-school children as vulnerable subgroup experiencing elevated distress early after COVID-19 outbreak [Moreover, most studies that report sex differences rely on more data from females than males, particularly when self-report measures or experience sampling methods were applied .Adverse life events are considered primary sources of risk for negative mental health outcomes including mental disorders, however, most individuals withstand, adapt to and facilitate recovery from adversity. Sex differences in resilience to stress have been reported, though most experimental work has been done in animals, indicating that while the behavioral outcome may be similar, the underlying neural circuits and operating resilience factors may not be concordant between the sexes . For exaIn line with this, more recent longitudinal studies and meta-analyses draw a more differential picture regarding population mental health during COVID-19, indicating that a vast majority of people is resilient , 8. Of nThe global pandemic can be positively used for mental health research and practice if it motivates us to work towards a more sex- and gender-sensitive research on mental health outcomes which considers and overcomes potential biases, including stereotypical and binary concepts, and at the same time, does not overlook specific needs of all included groups. This would ultimately also result in a better detection and recognition of mental health needs of and within the studied groups as many other factors interacting with biological sex and gender role will be assessed. By striving for this, we can improve our prevention and treatment efforts for the whole society.


Psychiatric disorders can affect and are even conditioned on our ability to successfully and enjoyably interact with other people. Conversely, facing difficulties in social relations or being socially isolated is known to increase the risk of developing a psychiatric disorder and strongly impacts symptom progression and health outcomes. This tight link between social interaction and risk for mental health challenges has been taken to suggest that psychiatric disorders can be construed as disorders of social interaction . This liThis Research Topic on “Social Interaction in Neuropsychiatry” attracted a sizable number of contributions from ~100 authors who addressed important questions about the nature of social interaction, its behavioral and neural mechanisms and relationship to psychiatric disorders. A total of 16 articles were published under this Topic, encompassed under four broad themes spanning clinical implications, developmental perspectives, contextual considerations on processing and methodological innovations.Blanchard et al. examined the ways in which sleep problems, commonly found in individuals with psychosis, lead to severe social impairments. Across clinical diagnoses, they evaluated multiple social domains, and found that addressing sleep problems can have a strong beneficial effect for improving both social skills and, indirectly, improving psychotic symptoms. This insight suggests a new avenue for approaching major neuropsychiatric symptoms, with an intervention that is relatively simple and without significant risk . Looking beyond the individual to the developmental dyad of mothers and their children, Apter-Levy et al. demonstrated that chronic depression alters mothers' dehydroepiandrosterone (DHEA) and DHEA-to-cortisol ratio. In turn, these alterations in hormonal levels drive reductions in sensitive maternal caregiving. However, as the authors point out, this is a delicate developmental “dance,” with improvements in maternal sensitivity eliciting from the child behaviors that further improve DHEA levels and balance DHEA-to-cortisol ratios, thus potentially initiating a positive social biofeedback mechanism. In a time at which many are carefully examining their own implicit biases towards others,  Hauschild et al. provide evidence for an own-age bias in facial emotion recognition for adolescents with and without autism spectrum disorder (ASD). This discovery has probably not been anticipated because in many fields autistic individuals would not have been predicted to be as susceptible to these common cognitive-perceptual biases. This is an intriguing new lead that deserves further study to understand the possible clinical implications. Finally, several systematic reviews appear in this research topic covering a range of both relatively mature, yet still exciting and growing areas of research. These include the role of speech prosody in psychopathology and linguistics  and empathic accuracy in clinical populations  to relatively new areas including the study of social cognition in obsessive-compulsive disorder .A key theme of many articles in this Research Topic pertains to how disruptions in or atypical social processing can contribute to risk for neuropsychiatric disorders. At the same time, working to address social processing impairments can offer uncharted opportunities for resilience in the face of neuropsychiatric conditions. White et al. provide a unique account of the neurobiological “embedding” of disordered social interactions, and how this may lay a path toward psychopathology in later life. They describe a functional neuro-anatomical model of typical and disordered human attachment (NAMA) which explains the emergence of a disorganized attachment style through either hyper- or hypo-arousing social interactions with caregivers, who act as either a threatening or insufficient source of co-regulation, respectively.Taking a developmental perspective, a trio of articles provide unique insights into how social interactive experiences during early childhood and adolescent years  may have important and lasting consequences for lifelong mental health. In their article “A Social Neuroscience Approach to Interpersonal Interaction in the Context of Disruption and Disorganization of Attachment,” Cataldo et al. address a timely issue of “Social Media Usage and the Development of Psychiatric Disorders in Childhood and Adolescence,” a topic that has gained particular relevance in the aftermath of the global lockdowns imposed during the COVID pandemic. Problematic social media use during the ages of 10 to 19 is shown to be linked to a variety of mental health issues including depression, anxiety, eating, and neurodevelopmental disorders. Finally, Blair et al. provide empirical evidence that sexual abuse during the adolescent years is associated with a heightened neurobiological response to threatening stimuli – including both faces and animals. This heightened neural responsiveness was observed in regions beyond the amygdala, including the frontal gyrus and posterior cingulate gyrus, suggestive of widespread and fundamental changes in the individual's basic perceptual and emotional processing. Collectively, these articles highlight the developmental sequelae of disordered social interactive experiences during the formative years, and their impact on the circuitry and organization of the developing brain.Furthering the developmental theme, Hernandez et al. examine the neural correlates of speech perception in the presence of ecologically-valid environmental noises in youth with and without autism spectrum disorder (ASD). Their findings suggest that a left-hemisphere language processing region may provide a compensatory mechanism in autism to attend to speech in the presence of competing background noise and potentially facilitate more successful social interactions.  Rolison et al. examine contextual effects on neural processing through a dual-brain EEG set-up. They demonstrate that the “resting brain” is different in the presence of a person, regardless of interaction status. Further, the extent to which one's social partner's physical orientation  modulated EEG gamma band power was related to self-reported social functioning. These findings underscore the importance of understanding the brain's “default mode” within the social context. The relatively novel methods discussed in this special issue also allow for a better understanding of real-world social perception.  Vettori et al. use a frequency tagging method to identify visual and neural preference for social (faces) compared to non-social (houses) stimuli presented within the same visual stream. By means of this method, the authors revealed a social bias in typically developing participants but no such bias in autistic participants.During real-world social interactions communicative information is embedded within a rich context that is complex, dynamic and often not directly observable. Social and nonsocial features of this complex environment interact to affect attention and neural processing. Further, distractors compete with the relevant social-communicative signals preventing effective social interaction. Thus, to understand both typical and atypical social interaction, social processes should be situated within the appropriate context. Two papers within this issue examine how context  affects neural processing and how that neural processing is closely related to social ability. Okruszek and Chrustowicz et al. provide a novel open-access database of point-light displays that focus on reciprocal actions of multiple agents which incorporates social interactions, emotions, and differing perspectives . This toolbox addresses the need for obtaining controlled stimuli depicting social gestures and bodily interactions with vast potential for application in neuropsychiatric research. Also, challenging the experimental designs in social isolation, the study by Rolison et al. implies that the mere presence of someone in the extrapersonal space may shape neural oscillations, suggesting that neural activity is tuned towards the presence of others.Understanding dysfunctional dynamics and mechanisms of social interaction requires methodological innovation at different levels that address experimental design and the overarching technical challenge of multi-subject measurement. A set of articles within this Research Topic highlight these important methodological challenges. Like faces, point-light walkers are frequently used as a measure of social perception. However, typical paradigms rely on a single person performing a non-communicative action . Pan and Cheng et al. provide an overview of current approaches to examine two-person interactions and discuss advances and challenges in moving the field towards more interactive settings. They distinguish eye-to-eye contact, body-to-body synchronization, and brain-to-brain coupling as central dimensions to summarize recent findings across clinical diagnoses outlining a novel perspective for two-person approaches in psychological interventions of psychiatric disorders. Addressing specific challenges of examining body-to-body interactions in the fMRI, Renvall et al., provide a proof-of-concept for measuring two interacting subjects within one MRI scanner. Their custom built fMRI coil allowed participants to lie face-to-face in a shared peripersonal space. The sufficient signal properties and the feasibility of this setup provide a perspective for more accessible means to characterize neural mechanisms of emerging phenomena during social interaction. A comparable technical challenge is to characterize dynamic, coordinated behavior under controlled and reproducible conditions, specifically in children. Here,  Baillin et al. use the “Human Dynamic Clamp”  to charTaken together, this unique collection of articles clearly demonstrates that social neuroscience and related fields of research have not only taken an “interactive turn” by focusing on the social interactive nature of human behavior , but thaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bemisia tabaci species complex (whitefly) causes enormous agricultural losses. These phloem-feeding insects induce feeding damage and transmit a wide range of dangerous plant viruses. Whiteflies colonize a broad range of plant species that appear to be poorly defended against these insects. Substantial research has begun to unravel how phloem feeders modulate plant processes, such as defense pathways, and the central roles of effector proteins, which are deposited into the plant along with the saliva during feeding. Here, we review the current literature on whitefly effectors in light of what is known about the effectors of phloem-feeding insects in general. Further analysis of these effectors may improve our understanding of how these insects establish compatible interactions with plants, whereas the subsequent identification of plant defense processes could lead to improved crop resistance to insects. We focus on the core concepts that define the effectors of phloem-feeding insects, such as the criteria used to identify candidate effectors in sequence-mining pipelines and screens used to analyze the potential roles of these effectors and their targets in planta. We discuss aspects of whitefly effector research that require further exploration, including where effectors localize when injected into plant tissues, whether the effectors target plant processes beyond defense pathways, and the properties of effectors in other insect excretions such as honeydew. Finally, we provide an overview of open issues and how they might be addressed.The  Bemisia tabaci  is a cryptic species complex consisting of at least 34 distinct genetic groups and 392 haplotypes  are thought to be among the most widespread and invasive B. tabaci species  form dense layers on the leaf surfaces that attract sooty molds and reduce photosynthesis  may reduce detection and defense induction by the plant host, thereby facilitating infestation. Upon feeding, a complex interaction between B. tabaci and the host plant occurs involving saliva and both electrical and hydraulic signals -dependent pathway and either suppress or do not measurably affect the expression of jasmonic acid (JA) and ethylene (ET)-regulated genes in Arabidopsis thaliana  genes that produce nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins that recognize pathogen effectors or their activities intracellularly. R genes can be further divided into the CC-domain-containing and TIR-domain-containing subfamilies  resistance to the pathogen. Conversely, the ability of plants to recognize effectors can depend on the presence of RLK/RLP receptor proteins or R proteins  to contribute to increased compatibility or are expected to do so (referred to as putative effectors). Effectors that were shown (in one or more plant–biotic interaction) to increase incompatibility are referred to as elicitors or avirulence factors.Pathogens and pests that successfully colonize plants have evolved mechanisms to overcome plant PTI and ETI. In plant pathology research, the word “effector” refers to “a B. tabaci and put these findings into the context of what is known about effectors from other phloem-feeding insects and plant-colonizing organisms. We will also critically discuss techniques used to identify and functionally characterize effector proteins and tools to identify and confirm their interacting partners.In the past decade, it has become clear that herbivorous arthropods produce effector molecules that modulate plant defense responses. Most studies of phloem-feeding insects have been performed with aphids and planthoppers, but several whitefly effector proteins were recently identified as well. The identification and functional analysis of these effectors is insect independent, as are studies of their modes of action and the identification of interacting plant proteins. This review will focus on effectors identified from B. tabaci, the primary salivary glands are located in the prothorax near the head and consist of at least 13 symmetrical cells. The accessory glands are located near the anterior part of the prothorax behind the primary salivary glands and consist of four cells. In both types of salivary glands, the cells contain microvilli lined into the central lumen of the gland. The gland lumens empty into primary or accessory salivary gland ducts, which are connected to each other   gene is upregulated in salivary glands when the secondary symbiont Serratia symbiotica is present  bacterial symbiont Portiera aleyrodidarum and one or more secondary (facultative) bacterial symbionts such as Hamiltonella, Wolbachia, and Rickettsia species. Portiera is vertically transmitted via the female line into the developing egg before it is laid, while the secondary symbionts may be both vertically and horizontally transmitted  and phytoalexins in both rice cells and seedlings and activate diterpene-based defense responses  in transcriptome data   holobiome. Plant proteins that functionally interact with such secondary effectors can be used for resistance breeding just the same since the insect's well-being is often strongly dependent on a stable interaction with their symbionts   decreases the survival rate of B. tabaci adults feeding on tomato plants but not on artificial diet. In addition, the expression of LAC1, both in salivary glands and the midgut, increases when whiteflies feed on JA-treated plants compared to control plants. Taken together with the evidence of LAC1 secretion in the artificial diet, this suggests that LAC1 helps B. tabaci to overcome plant defense responses and may act as effector in the plant cell , which was identified in the salivary gland transcriptome of B. tabaci MED based on their capacity to suppress the HR caused by Pseudomonas syringae pv. tabaci or P. syringae pv. syringae  production. Reactive oxygen species signaling is an important mechanism used by plants against phloem feeders and other insect herbivores   effector Bsp9 was identified by comparing the transcriptomes of whiteflies with and without TYLCV  assays. The Bsp9–WRKY33 interaction prevents WRKY33 from localizing to the nucleus , whereas this response is actually stronger induced in the presence of the effectors Bsp1 and Bsp5. These effectors were not analyzed further in this study, but additional analysis could provide more insight into DAMP-triggered responses by the host against whiteflies. Bsp9 is highly conserved in both the MEAM1 and MED.The B. tabaci (MED) salivary glands  protein extracts. In planta expression of Bt56 in Nicotiana tabacum increases the insect's survival and fecundity, while knockdown of this effector gene by RNAi in both N. tabacum and G. hirsutum decreases the performance of whitefly  transcription factor NTH202 in punctate structures in tobacco cytoplasm, as visualized by BiFC. This localization suggests that, like Bsp9, Bt56 is retaining a transcription factor from moving to the nucleus, preventing its function. Some SA- and JA-pathway genes are regulated by KNOX1 in maize. However, Xu and co-workers were careful to suggest that the altered SA levels caused by Bt56 were a direct result of this interaction, since SA levels in N. tabacum did not significantly change when NTH202 expression was silenced, but whitefly performance was improved  can be used to deliver effectors into plant cells such as tomato cells. This system was used to show that Me10 increases M. euphorbiae fecundity on tomato , and PIntO2  in A. thaliana, which all leads to increased aphid performance  or silencing the putative effector and perform fecundity or nymphal development assays which could give us insights into effectors needed for initial infestation or development. Finally, the transcript levels of effector genes may also differ depending on microbes present in the insects, as observed in the citrus psyllid D. citri; several effector genes were differentially expressed in adults and nymphs following infection with Candidatus Liberibacter asiaticus (Ca. Las)  is a posttranscriptional gene-silencing mechanism that is triggered by the presence of double-stranded RNA (dsRNA) in the cell  or chlorosis (Mp10)  suppresses ROS production and Ca2+ response induced by the bacterial elicitor flg22  suppress this response  identified a catalase that functions as an ROS scavenger to inhibit ROS burst  and threonine deaminase 2 (TD2) but not lipoxygenase D (LoxD). Taken together, these findings indicate that the whitefly effectors identified to date play various roles in manipulating hormonal pathways. We expect that in the near future, many more whitefly effectors involved in suppressing the immune response will be identified, and their exact roles and the underlying mechanisms will be further uncovered.An additional approach to identifying the roles of effectors in plant defense is to analyze hormonal differences. Effectors can alter the expression of phytohormone-related marker genes, and effector genes can be upregulated when an insect feeds on plants treated with phytohormones. The whitefly effector Bt56 increases the expression of the SA marker gene encoding pathogenesis-related protein 1a (PR-1a) in in planta. The yeast-two hybrid (Y2H) system is a relatively easy tool to identify possible host target proteins for an effector. Several target proteins of phloem-feeding insect effectors have been identified using this method . For this, plant tissue expressing a tagged effector of interest is used to pull out its plant target proteins that are subsequently analyzed by MS. This method also has several disadvantages. For example, a weak interaction could be disrupted during the washing steps, or rupture of the cells during protein extraction could allow proteins that are normally located in different cellular compartments to come into contact with one another including the effector  BiFC, where the effector and target proteins are fused to complementary halves of a yellow fluorescent protein (YFP), producing a YFP-fluorescent signal upon interaction; (ii) Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM), whereby energy transfer taking place between a donor and an acceptor chromophore when the two fused proteins interact is detected by fluorescence microscopy; (iii) Co-Immunoprecipitation (Co-IP), where the effector and host protein are expressed with different tags and the pull-down of one of these proteins results in the pull-down of the interacting protein as well, detectable with immunoblot analysis. One advantage of BiFC over Co-IP is that it is relatively easy to perform and weak interactions are also visible using BiFC. In addition, BiFC and FRET-FLIM reveals where the interaction takes place within the plant cell as was shown for the orthologs Bsp9 and Bt56 with WRKY and NTH202, respectively, which both occur in the cytoplasm . Whereas, these studies have provided information about large-scale evolutionary processes, such as chromosome organization, comparisons at this scale may be less useful for analyzing more recent evolutionary events involving effector genes. Hence, future research may focus on comparative genome analyses of closely related species with different plant host preferences. The B. tabaci species complex is a good candidate for this type of analysis, as there are many species with known host specificity  via EMS (Ethyl Methane Sulfonate) or CRISPR-based mutagenesis. Both approaches require a thorough understanding of the interaction domain of the plant protein and the effect of mutations in this domain on the phenotype of the plant. This would require complementation studies in which mutated forms of the plant protein are expressed in knock-out plants.The plant interactors for some effector proteins were identified, providing more detailed insight into what these effectors accomplish in the plant cell. Altering the expression level of the corresponding plant genes leads to moderately altered levels of resistance. This incomplete or partial level of resistance phenotype indicates that we are dealing with a polygenic trait (Kliebenstein, in planta. Although answering these questions will truly be challenging, several fundamental questions eventually need to be addressed to understand the functions of effectors in planta: In which cells are they active? Does this coincide with the cells in which the interactors are expressed? Are the effectors systemically transported? In order to better select effectors relevant to phloem-feeding insects, we need assays that are located in the phloem. The agroinfiltration assays described above have primarily involved the transformation of epidermal and mesophyll cells. One possible option is to adapt the phloem-localized GCaMP3 fluorescent protein-based [Ca2+]cyt sensor, which reports increased [Ca2+]cyt upon herbivory in A. thaliana, for use in the model plant of choice (Vincent et al., Other outstanding questions involve the localization of the effector proteins in planta, and how can we use this knowledge to increase plant resistance to whiteflies?We hope that the field of insect-effector biology will grow in order to achieve the critical mass needed to study these topics in detail. Finally, the discovery that RNA molecules from insects, including sRNAs and long non-coding RNAs, are transported into plants has opened up a whole new field of research. However, the questions about these molecules also revolve around a central theme: What is their mode of action DN and RS conceived the manuscript. SD, PK, DN, and RS conceived the figure. SD made and further designed the figure. MM checked and updated the supplementary data. RS and SH critically revised the manuscript. PK extensively rewrote the manuscript. All authors contributed to writing and discussions on the content and read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro–adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis. Cilia are evolutionarily conserved, microtubule-based cellular appendages that perform numerous functions required for normal development. Ciliary dysfunction underlies a broad spectrum of clinical conditions collectively known as ciliopathies. Cilia are composed of a core of microtubule bundles, termed axonemes, that are ensheathed in a ciliary membrane compartment and anchored at the base by the matured mother centriole or basal body A. In somSkeletal muscle is comprised of striated, multinucleated myofibers organised into compact bundles. During development, myogenesis is initiated when multi-potent mesodermal cells are committed to skeletal myoblasts. These myoblasts are muscle precursors that express Pax3 and Pax7 and exhibit a high proliferative capacity. Following a proliferative phase, the expression of the muscle regulatory family (MRF) of transcription factor proteins  coincides with cell cycle arrest, cessation of myoblast proliferation and initiation of differentiation, accompanied by alignment of the mononucleated myoblasts and their fusion to form multinucleated syncytia called myotubes that assemble contractile cytoskeletal units, known as sarcomeres C. These +ve), also referred to as satellite cells, are retained in a quiescent state and are located outside the myofiber lying between the sarcolemma and the external lamina ; these cells initiate proliferation and generate myoblasts that are principally responsible for the regenerative capacity of skeletal muscle. When activated, some satellite cells undergo oriented asymmetric divisions to give rise to muscle-committed progenitors (myoblasts) and a self-renewed ‘stem cell’ population that remains in the ‘satellite’ position on the myofiber surface ,In primary myoblast cultures, following serum withdrawal, ciliogenesis coincides with cell cycle exit and the early onset of differentiation C 15]. T. T15]. T+ve myoblasts but are absent in myofibers  and differentiating (Myogenin+ve) cells in normal adult skeletal muscle , a common paediatric tumour that histologically resembles embryonic skeletal muscle and is thought to arise from a defect in myoblast differentiation and an arrested state of skeletal muscle development ,60. The To date, the specific description of the pathological consequences of satellite cell cilia dysfunction has been limited largely to RMS. The concept of some primary intrinsic dysfunction of satellite cells has been proposed for various complex neuromuscular diseases , without consideration of cilia, although it seems likely that possible altered myogenesis may instead more widely reflect interactions with an adverse ECM (as discussed below). Nevertheless, the extent that disrupted cilia regulation of satellite cell fate might contribute to common myopathy states more broadly warrants investigation. Moreover, in the context of cilia on satellite cells/myoblasts and neuromuscular diseases, it is relevant to consider the wider impact that some drugs might have on cilial health, especially microtubule-targeting drugs used in chemotherapies.The fusion of myoblasts to generate multi-nucleated myotubes that mature into myofibers is critically required for normal muscle development and regeneration ,62,63. TAs mentioned previously, cilia are present on fate-committed myoblasts but are disassembled during myoblast fusion. This occurs in concert with the reduction in centrosomes and the reorganisation of the microtubule and actin cytoskeleton necessary for the formation of multi-nucleated myotubes and sarcomere assembly. It is proposed that γ-actin plays a key early role in initiating sarcomere assembly of sarcomeric proteins during differentiation . InteresThe disassembly of cilia during myoblast fusion suggests that ciliary functions may not be required for this stage of myogenesis. However, our close inspection of differentiating cultured myoblasts indicates that remnants of the ciliary membrane, as marked by Arl13b, attached to centrioles are clearly visible within multi-nucleated myotubes . MoreoveIn combination with intrinsic factors, the extrinsic ECM environment in skeletal muscle is an important determinant of adequate myogenesis for development and regeneration . SkeletaThe excessive fibrosis and pathological accumulation of ECM proteins observed in ‘ciliopathies’, such as renal cystic and Bardet-Biedl syndromes, suggests a proper cilia–ECM dialog is important for normal cellular functions and organ development. The ECM influences cilia length and ECM components, such as laminin, and collagen has been shown to promote cilia growth ,75. TherIn addition to sensing the extracellular environment, ciliary signalling in satellite cells and differentiated myoblasts may also contribute to remodelling of the ECM. Hedgehog signalling in chondrocytes brings about changes in ECM composition associated with bone development . SimilarMyogenic stem cells may regulate ECM composition in a cell-autonomous or non-cell autonomous fashion. It is well established that FAPs/fibroblasts and myofibroblasts represent the main protagonists in fibrosis of skeletal muscle ,81 and oIn skeletal muscle tissue, primary cilia are clearly present on skeletal muscle progenitors, quiescent and activated satellite cells and myoblasts and differentiating myoblasts (but not on proliferating myoblasts), and on fibroblasts/FAPs and other interstitial cells, as well as cells associated with blood vessels and nerves. The important functions of cilia in cell cycle regulation and cell fate specification are now evident, and emerging evidence hints at undescribed functions in myoblast fusion and the regulation of ECM organisation in the skeletal muscle microenvironment surrounding myofibers and satellite cells. A complete picture of cilia function in skeletal muscle health and disease is yet to fully emerge. To date, no studies have yet been attempted to specifically ablate cilia in satellite cells, and the feedback interactions between cilia on various cell populations in skeletal muscle tissue require more extensive interrogation. Finally, the intrinsic contribution of cilia in satellite cells/myoblasts or extrinsic roles in non-muscle cells within the context or normal ageing muscles and sarcopenia and neuromuscular diseases, such as muscular dystrophies, remains undefined. It is likely that more consideration of the contribution of cilia in these situations and a better delineation of their roles related to modulation and interactions between myogenesis and the ECM may lead to an improved understanding of disease mechanisms or novel treatment strategies.


The new coronavirus disease 2019 pandemic has spread throughout most of the world. Cerebral venous thrombosis is a rare thromboembolic disease that can present as an extrapulmonary complication in coronavirus disease 2019 infection.We report the case of a Hispanic woman with Down syndrome who has coronavirus disease 2019 and presents as a complication extensive cerebral venous thrombosis..Cerebral venous thrombosis is a rare thromboembolic disease that can present as an extrapulmonary complication in coronavirus disease 2019 infection. In the absence of clinical and epidemiological data, it is important to carry out further investigation of the risk factors and pathophysiological causes related to the development of cerebrovascular thrombotic events in patients with Down syndrome with coronavirus disease 2019 infection The new coronavirus disease 2019 (COVID-19) pandemic has spread throughout most of the world, causing more than 28 million infections to date, with more than 1 million deaths worldwide  and magnetic resonance venography [OR 38.34 ] have a high level of diagnostic precision in cerebral venous thrombosis [Regarding the diagnostic imaging performed, magnetic resonance imaging (MRI) was not considered since the diagnosis could be made with simple chest scan and angiotomography; simple axial tomography can show specific signs of cerebral venous thrombosis in up to 30% of cases, such as the dense triangle sign and the cord sign. If the diagnosis is suspected, it should be confirmed with computed tomography plus venography, which can show absence of partial or complete flow in thrombosed veins or sinuses . The guirombosis .et al., our patient had a score of 6 for a 30-day mortality risk of 10%; however, the outcome presented in the patient at 3-month follow-up from the onset of symptoms showed no complications or sequelae related to cerebral venous thrombosis, or fatal outcomes [In terms of the management of cerebral venous thrombosis, anticoagulation is the treatment of choice as soon as the diagnosis is made. When using low-molecular-weight heparin and unfractionated heparin, no significant difference was found with respect to bleeding, mortality, and morbidity outcomes . Accordioutcomes .In conclusion, cerebral venous thrombosis is an infrequent thromboembolic disease, presented as an extrapulmonary complication in COVID-19 infection, which manifests itself mainly in fertile women, with headache being the most common symptom. Diagnosis can be made by computed tomography plus venography or magnetic resonance venography. Currently, there are no epidemiological data or pathophysiological causes related to the development of cerebral venous thrombosis in patients with Down syndrome with COVID-19 infection.