Integrating data on the architecture, kinetics, and transcriptional input–output function is a promising approach to uncovering the underlying dynamic mechanism. For eukaryotes, distinct bursting features described by the multi‐scale and continuum models coincide with those predicted by four theoretically derived principles that govern how the transcription apparatus operates dynamically. This consistency suggests a unified framework for comprehending bursting dynamics at the level of the structural and kinetic basis of transcription. Moreover, the existing models can be unified by a generic model. Remarkably, transcriptional bursting enables regulatory information to be transmitted in a digital manner, with the burst frequency representing the strength of regulatory signals. Such a mode guarantees high fidelity for precise transcriptional regulation and also provides sufficient randomness for realizing cellular heterogeneity.There is accumulating evidence that, from bacteria to mammalian cells, messenger RNAs (mRNAs) are produced in intermittent bursts – a much ‘noisier’ process than traditionally thought. Based on quantitative measurements at individual promoters, diverse phenomenological models have been proposed for transcriptional bursting. Nevertheless, the underlying molecular mechanisms and significance for cellular signalling remain elusive. Here, we review recent progress, address the above issues and illuminate our viewpoints with simulation results. Despite being widely used in modelling and in interpreting experimental data, the traditional two‐state model is far from adequate to describe or infer the molecular basis and stochastic principles of transcription. In bacteria, DNA supercoiling contributes to the bursting of those genes that express at high levels and are topologically constrained in short loops; moreover, low‐affinity  I.et al., et al., et al., et al., et al., et al., et al., et al., Like a coin with two opposite sides, gene transcription exhibits duality. On the one hand, it is an accurate process. When and at what rate to transcribe a gene is subject to precise regulation, and accuracy is essential for cell fitness and development  was believed to fluctuate slightly around an average , it was observed that a number of mRNAs from a gene emerge suddenly and then disappear gradually during a short time window , inactivation rate koff (reaction rate from ON to OFF), and mRNA synthesis rate from the ON state km. kon is positively related to the concentration of transcriptional activators. Both koff and km are constant, i.e. the duration of the ON state is exponentially distributed and mRNA synthesis is a Poisson process.Intuitively, transcriptional bursting can be characterized with a simple two‐state model Fig. A, where et al., The original two‐state model was proposed to explain cell heterogeneity 25 years ago Ko, . It is wIII.et al., via tracing the time series of transcription initiation events or changes in MS2/PP7‐labelled mRNAs. While this is realizable, it has not been done sufficiently. Nevertheless, it was confirmed that mRNA production is not a Poisson process but can be well fitted by the two‐state model , the bursting was revealed to be primarily caused by DNA supercoiling . In contrast with the traditional view that transcription initiation is stimulated by an NtrC hexamer simultaneously bound to the two enhancers, Wang et al. . With the DNA bridging, the NtrC hexamer at the distal enhancer is in the vicinity of the TTS and thus immediately stimulates the polymerases one by one, leading to a burst. This work stresses the importance of low‐affinity sites, unstable protein complexes, and transient molecular interactions in orchestrating transcription.Upstream of the g et al.  revealedg et al. . MoreoveIV.et al., In higher eukaryotes, DNA is packaged by nucleosomes that consist of histones; both the DNA and histones are epigenetically modified , then the open complex (OPC), and the elongation complex, which synthesises a transcript  round of cycling and T is the total time corresponding to n rounds of cycling. As the activator concentration rises, n becomes larger in probability for fixed T, and so does RTOR. In this way, RTOR robustly encodes the activator concentration ’ between the enhancer and the Mediator Fig. A, which ion Fig. B. Note tThe Mediator transfers information from the settled activator to Pol II through allostery. The entry of an activator into the C‐space induces allostery in the Mediator, facilitating Pol II to assemble and initiate transcription. Without the activator, transcription initiation rarely occurs.RTOR is converted to the number of transcripts.With an activator in the C‐space, a convoy of Pol IIs is launched, leading to a burst of transcripts. The time interval between two successive initiation events is far shorter than the activator's residence time. Typically, the interval is of seconds. Such intensive initiations together with the Mediator's allostery guarantee that the basic unit of Based on the general structural organization and conformational changes of the TA, the ensemble and probability theories were exploited to probe how the TA dynamically operates  model Fig. E, with wres Fig. . AdditioVI.et al., et al., et al., et al., RTOR is exploited by the TA to encode activator concentration and guide transcription initiation  Genes are transcribed in episodic bursts. This feature is ubiquitous from bacteria to mammalian cells, regardless of whether they are constitutive or inducible genes.(2) Transcriptional bursts in eukaryotes take place with multiple timescales, and the transcription initiation rate is continuous during each ON period. Such features are consistent with those dictated by four theoretically derived principles that govern how the TA operates dynamically. This in turn suggests that eukaryotic genes likely share similar dynamic principles for TA operation, as they share the same set of general transcription factors.(3) The ratchet, multi‐scale, and continuum models emphasize different profiles of transcriptional dynamics. These models are applicable to genes of specific categories and can be unified by the WLW model, which depicts the core steps of regulated transcription and can be accommodated to characterize gene specificity. Additionally, conclusions derived from the traditional two‐state model should be treated cautiously.(4) Transcriptional bursting in eukaryotes allows digital information conversion, by which regulatory signals modulate the burst frequency rather than burst size. For a given TA, the burst size obeys a specific distribution; experimentally, the burst size might be mistaken as subject to modulation since it is hard to differentiate a burst from a burst cluster.RTOR code, which represents an accurate encoding of time‐varying activator concentration; moreover, bursting‐induced fluctuations in mRNA number can be smoothed by nuclear retention of mRNAs. On the other hand, such fluctuations can also be enlarged to achieve sufficient heterogeneity.(5) Transcriptional bursting fulfills the accuracy and stochasticity requirements for the transcriptional response. The modulation of burst frequency is an embodiment of the et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., (6) Low‐affinity DNA binding sites, unstable protein complexes, and DNA supercoiling can play crucial roles in regulating transcription. Investigating transcriptional dynamics necessitates both live imaging methods with high resolution  The significance of various bursting patterns from different promoters remains to be addressed. Firstly, unscrambling the bursting patterns paves the way to reveal the dynamics of both the chromatin and the TA. By statistically analysing the Mediator‐ and TBP‐dependent bursting dynamics , for example, it can be inferred that the Mediator interacts with the DNA much more transiently than the TBP (Tantale VIII.This work was supported by the Ministry of Science and Technology of the People's Republic of China [2013CB834104], National Natural Science Foundation of China , and Fundamental Research Funds for the Central Universities [JUSRP11838].


Dear editor, “Lateral approach versus combined lateral and anteromedial approach for surgical treatment of terrible triad of the elbow: A meta-analysis” by Meena et al., [We read with great interest the article et al., . They caet al., [et al., [et al., [p=0.03) and fewer number of complications (p=0.02). So, the conclusion of the meta-analysis is questionable. The new conclusion that can be drawn is that majority of TTIE can be operated by the lateral only approach due to shorter time duration and fewer complications with no difference in the functional outcomes between the two approaches.Our first observation is that authors included four studies for this meta-analysis, of which study by Li et al.,  is serie[et al.,  and Liu [et al., , both coNone declared.


Strizova et al.'s  recent rBorrelia burgdorferi has been shown to manipulate both the innate and adaptive immunity of its mammalian hosts in a variety of ways (Tracy and Baumgarth Borrelia burgdorferi. The organism abrogates the development of highly specific long-term immunity to persist in the host (Elsner et al. In short, the persistence of IgM antibodies in Lyme disease may represent chronicity, not a false positive. The classical pattern of B cell maturation and the IgM to IgG transition are lacking in chronic infection by


P. falciparum erythrocyte invasion. This review summarizes the current understanding of the molecular interactions that lead to rosette formation, with a particular focus on host uninfected erythrocyte receptors including the A and B blood group trisaccharides, complement receptor one, heparan sulphate, glycophorin A and glycophorin C. There is strong evidence supporting blood group A trisaccharides as rosetting receptors, but evidence for other molecules is incomplete and requires further study. It is likely that additional host erythrocyte rosetting receptors remain to be discovered. A rosette-disrupting low anti-coagulant heparin derivative is being investigated as an adjunctive therapy for severe malaria, and further research into the receptor–ligand interactions underlying rosetting may reveal additional therapeutic approaches to reduce the unacceptably high mortality rate of severe malaria.Malaria remains a major cause of mortality in African children, with no adjunctive treatments currently available to ameliorate the severe clinical forms of the disease. Rosetting, the adhesion of infected erythrocytes (IEs) to uninfected erythrocytes, is a parasite phenotype strongly associated with severe malaria, and hence is a potential therapeutic target. However, the molecular mechanisms of rosetting are complex and involve multiple distinct receptor–ligand interactions, with some similarities to the diverse pathways involved in  Plasmodium falciparum infected erythrocyte (IE) adhesion phenotype that is associated with severe malaria in sub-Saharan Africa  as rosette-mediating adhesion molecules  results in smaller, weaker rosettes, equivalent to those seen in group O erythrocytes  that map to the C3b binding site on CR1 being effective inhibitors  are found on cell surfaces and in the extracellular matrix of many tissues, and have a role in multiple aspects of the et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Fluorescently-labelled heparin does bind to the surface of erythrocytes infected with rosetting parasites more than non-rosetting lines   was partially inhibited by a GYPC mAb (clone Ret40f) and by soluble recombinant GYPC , and was based on a single count for each isolate with no replication . Plasmodium falciparum isolates were not tested with the GYPC knockdown erythrocytes.For et al., P. falciparum culture-adapted parasite lines, Rowe et al. . Gerbich-negative erythrocytes formed rosettes normally with the five parasite lines tested. Goel et al. also report normal rosetting of Gerbich-negative erythrocytes from two donors (Goel et al., If GYPC is a rosetting receptor, it is possible that the ‘Gerbich-negative’ blood group type, which is common in Melanesian populations (Patel e et al.  tested tP. falciparum and P. vivax rosetting.Taking into account all existing evidence, further investigation of a wider range of parasite lines is needed to determine whether GYPC is an important host receptor for both P. falciparum erythrocyte invasion (Sim et al., et al., et al., GYPA is a highly-expressed erythrocyte surface glycoprotein that carries the MNS blood group antigens. It is known to be a receptor for et al., et al., There is some limited evidence to suggest that GYPA may have a role in rosetting. Parasites of the strain FCR3S1.2 transfected with a specific RIFIN gene formed rosettes that were largely dependent on blood group A (Goel P. falciparum and 11 P. vivax clinical isolates (Lee et al., et al., P. falciparum lines (Rowe et al., Despite the above positive evidence, there are no other data supporting a role for GYPA in rosetting. GYPA mAb fragments had no inhibitory effect on rosetting in ten et al., et al., None of the receptors described above fully account for the adhesion interactions between infected and uninfected erythrocytes, and it is likely that other host rosetting receptors remain to be identified. There is evidence to suggest that these unknown host receptors are carbohydrates or protease-resistant proteins, because uninfected group O erythrocytes treated with trypsin and other proteases are still able to form rosettes (Udomsangpetch et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., In order to progress rosetting research, alternative methods are needed. Rosetting experiments with GYPC and CR1 knockdown cultured human red cells derived from CD34+ haematopoetic stem cells have been performed (Lee Of the rosetting receptors described over the past 30 years, only the blood group A trisaccharide has been authenticated by a variety of methodological approaches from a range of different investigators. For all other potential rosetting receptors, the evidence remains fragmentary  and furt


The application of engineering principles such as standardisation, together with several key technical advances, enabled a revolution in the speed and accuracy of genetic manipulation. Combined with mathematical and statistical modelling, this has improved the predictability of engineering biological systems of which nonlinearity and stochasticity are intrinsic features leading to remarkable achievements in biotechnology as well as novel insights into biological function. In the past decade, there has been slow but steady progress in establishing foundations for synthetic biology in plant systems. Recently, this has enabled model‐informed rational design to be successfully applied to the engineering of plant gene regulation and metabolism. Synthetic biology is now poised to transform the potential of plant biotechnology. However, reaching full potential will require conscious adjustments to the skillsets and mind sets of plant scientists.Powered by developments that enabled genome‐scale investigations, systems biology emerged as a field aiming to understand how phenotypes emerge from network functions. These advances fuelled a new engineering discipline focussed on synthetic reconstructions of complex biological systems with the goal of predictable rational design and control. Initially, progress in the nascent field of synthetic biology was slow due to the  Perhaps the most visible aspects have been impactful tools and techniques such as large‐scale DNA assembly, genome engineering and cell‐free protein expression. Thus, there has been a tendency for biologists to perceive synthetic biology as a field focussed on the development and application of novel technologies, while the media typically report it as ‘advanced biotechnology’. However, synthetic biology self‐defines as an emerging discipline of engineering. At its core is the central theory that applying engineering principles and approaches improves the predictability and increases the ease and efficiency with which biological systems can be designed, constructed, and characterised . These approaches are applied in iterative cycles of design–build–test–learn, where quantitative data on performance are fed into predictive models to improve later design cycles Fig. . The appet al., It should be noted, however, that there is not total acceptance that synthetic biology is able to operate as a field of engineering Kwok, . Engineest century .Plant synthetic biology encompasses diverse research areas, each focussed on improving the predictability of a particular approach to reprogramming biological systems through the application of engineering principles Fig. . These aife Fig. . These ret al., et al., Recent reviews have examined how crop‐improvement or the production of biofuels and plant natural products might be advanced by applying synthetic biology approaches  improved efficiency and flexibility. However, the recombinase‐enabled reaction did not allow for complex or precision design. Further, the proprietary nature was undesirable in the emerging synthetic biology community, which, inspired by progress in electronic and software engineering, prescribed that standards, infrastructure and enabling technologies should be ‘open’ in order to accelerate progress and provide an ecosystem encouraging of innovation and entrepreneurship  play an important role in natural gene regulatory circuits and artificial miRNAs (amiRNA) are widely used for silencing target genes  have been engineered into bacterial, yeast and mammalian cells , those beyond the canonical 20 used by the majority of organisms, have been used to control post‐transcriptional modifications and to enable site specific labelling for visualisation in living cells, as well for the production of antibody drug‐conjugates  are used to investigate intracellular metabolic reactions and to study biological behaviours (Miller & Gulbis, et al., The engineering of biologically derived materials into useful products sits at the cusp of synthetic biology and biomaterials. As synthetic biology approaches are being used to engineer biomolecules and materials, convergence could lead to new developments in the control of tissue organisation, the understanding of which is a major goal in fundamental biology and essential for progress in regenerative biology (Keating & Young, et al., et al., et al., et al., Mycoplasma genitalium genome with 52 fewer genes (Hutchison et al., E. coli genome that uses just 59 codons to encode 20 amino acids (Fredens et al., et al., Synthetic genomics, the bottom‐up redesign of genomes, is often considered as the ultimate exemplification of synthetic biology. The first genome‐scale DNA assembly project was completed in 2008 (Gibson These emerging research areas are developing exciting opportunities for plant science. Alongside advances in knowledge and novel technologies, these projects also provide the potential of new roles for plants in human life and society.VII.Marchantia polymorpha, offer advantages for the systems‐scale experiments that aid synthetic biology. Able to complete its lifecycle within a Petri dish, it is possible to track physiological and morphogenetic changes through the entire life cycle. Comparatively low gene numbers mean that functional studies of gene families, reconstruction of transcriptional networks, and the development of genome‐scale models are simplified (Boehm et al., et al., et al., et al., et al., The ability to engineer predictably is hampered by the unknown. Iterative progressions of design–build–test–learn cycles are useful not only for optimising the performance of synthetic systems but also for improving models of biology. Synthetic biology approaches are therefore as relevant to fundamental plant biology as they are to plant biotechnology. However, many of the approaches discussed above remain challenging in long‐lived plant species with low transformation efficiencies. The use of modelling and streamlining experiments through, for example, the application of DoE, can be used to focus engineering efforts. However, more tractable species are better suited for complex manipulations. The numerous genome‐scale datasets and resources available for Arabidopsis underpin its importance in plant science, but simpler species such as the liverwort, et al., et al., et al., et al., et al., et al., et al., Synthetic biology approaches are being applied to numerous applications of plant biotechnology. In this review there are examples of how synthetic biology is being applied to increase crop yields (South et al., et al., The strength of synthetic biology lies in its intellectual diversity. While the majority of presenters at a typical plant science meeting will have trained as biologists, at synthetic biology meetings mathematicians, computer scientists, engineers, biologists, chemists and physicists will all be present in varying proportions. Arguably, the most thrilling achievements have been made at the overlaps of traditional disciplines (Cameron


Secondary fracture healing is a complex multi‐stage process in which the mechanical environment plays a key role. The use of an appropriate mechanical stimulation such as strain is conducive to tissue formation between fracture ends, thus aiding the healing process. However, if the strain is too large or too small, the biological behavior of the cells involved in bone healing will be affected, resulting in non‐union or delayed healing. In this review, we summarize the current state of knowledge regarding the effect of strain on cells that play a role in the fracture‐healing process. Overall, the related literature suggests that selection of an adequate strain promotes fracture healing through the stimulation of angiogenesis and osteogenesis, along with inhibition of osteoclast differentiation and bone resorption. However, standardized methods for the application of mechanical stimulation are lacking, and a unified consensus on the mechanism by which strain promotes cell differentiation has not yet been reached. These issues, therefore, deserve further investigation. This article summarizes the state of knowledge regarding the effect of strain on cells involved in the fracture‐healing process. In fact, mechanical stimulation regulates the process of osteogenic differentiation. Osteoblasts are mainly derived from MSCs; they can continue to differentiate into bone cells and are embedded in the bone mineralized matrix. Osteoclasts are derived from hematopoietic stem cells under the influence of mechanical factors; they differentiate from monocytes into multinucleated cells and perform the function of bone resorption. Importantly, different types of strain will have different effects on these cellsFracture healing is a complex process that can be divided into the following stages: hematoma, granulation tissue formation, callus formation, and bone remodeling. The healing process is carried out by specific key cells including inflammatory cells, endothelial cells, fibroblasts, mesenchymal stem cells (MSCs), osteoblasts, and osteoclasts; not only the cells but also their secreted substances are importantvia external skeletal fixation to establish a fracture model. Importantly, the healing process was compared between a control group in which rigid fixation was maintained, and an experimental group in which mechanical stimulation  was applied daily. The authors found that the level of bone callus formation and the stiffness of the fracture site measured both in vivo and in vitro were higher in the experimental group than in the control group. Using the same model of tibial fracture, Kenwright et al.Goodship and KenwrightThis article reviews the available literature on the effect of applied strain in the proliferation, differentiation, and activity of different types of cells involved in fracture healing. This review aims to deepen the understanding of the fracture healing process and provide a reference for appropriate fracture treatments.et al.et al.et al.MSCs migrate to the fracture site from the surrounding tissues and blood circulationet al.When a fracture occurs, activated platelets release a variety of products including fibronectin (FN), platelet‐derived growth factor (PDGF), and transforming growth factor‐β (TGF‐β). These substances trigger an inflammatory response, allowing inflammatory cells including fibroblasts and endothelial cells to enter the fracture gap and form new blood vessels and granulation tissueet al.Blood vessel formation after a fracture can be induced by a variety of cytokines, among which vascular endothelial growth factor (VEGF) plays the most critical roleTherefore, mechanical stimulation can enhance the angiogenesis of early hematomas, and VEGFR2 seems to play an important role in this process, which can persist even after removal of the mechanical stimulation.et al.et al.et al.The fibroblasts involved in fracture healing are mainly derived from the endosteum and periosteumet al.Yamamoto In addition, fibroblast growth factor (FGF) can promote the osteogenic differentiation of fibroblastsDifferent mechanical environments have different effects on MSC differentiation. Chen and Jacobset al.et al.et al.et al.Haasper et al.Sox9 and Col2 showed a gradual upward trend with increasing time of mechanical stimulation, and both markers were significantly higher than those in the control group without mechanical stimulation. Angele et al.Xie Collectively, these studies indicate that appropriate mechanical stimulation can promote chondrogenic MSC differentiation, which may be related to the RhoA/ROCK‐1 signaling pathwayet al.et al.et al.et al.Proliferation and differentiation of osteoblasts are regulated by various factors such as hormones, growth factors, and mechanical stimuliet al.In addition, Weyts Osteoclasts are cells with bone matrix resorption functionet al.et al.RANK, whereas high‐strain conditions produced the opposite result. Rubin et al.Odf mRNA in the experimental group was only 59% ± 3% that in the control group. In summary, proper mechanical stimulation can inhibit osteoclast differentiation and bone resorption.Studies have shown that the development and activity of osteoclasts are regulated by osteoblastsIn conclusion, recent studies have confirmed that most cells involved in fracture healing can respond directly or indirectly to mechanical stimulation and that the use of adequate mechanical stimulation is conducive to proper fracture healing. First, mechanical stimulation promotes the migration of MSCs and angiogenesis of early hematomas. Second, mechanical stimulation can promote the osteogenic differentiation of fibroblasts to aid the process of fracture healing. Third, adequate mechanical stimulation inhibits osteoclast differentiation and bone resorption, thus promoting bone reconstruction.However, the effect of mechanical stimulation still depends on the loading method used. Thus, similar mechanical stimulations may cause different or even opposite cell responses. The reason may be that the same mechanical transduction pathway may share different mechanical stimuli and that different signal pathways may interact with each otherAll authors listed meet the authorship criteria according to the latest guidelines of the International Committee of Medical Journal Editors, and all authors are in agreement with the manuscript.


Dear editor,We would like to thanks the readers of the Bulletin of Emergency And Trauma (BEAT) for their valuable comments regarding our recently published article. We appreciate their interest in our article . We ackn“There was no difference in Mayo elbow performance score (MEPS) between LA and CML groups ”.Another concern by the reader was the exclusion of two studies from the meta-analysis , 3. We det al., [1. The study by Pierrart et al.,  was not et al., [2. The study by Liu et al.,  was not We assume that further high quality randomized controlled trials (RCTs) are required to support our conclusion.None declared.


We thank Noud van Helmond and colleagues  for the In a number of studies, we have recently evaluated the possible value of a handy device (Viatom’s Checkme) for measuring multiple vital parameters, including cuffless blood pressure (BP) determination.The Checkme device entered the medical domain after it was originally designed for the consumer market. This makes it very interesting and necessary to scientifically investigate its use in patients.After a comparison with a common BP monitor  and an eWe are aware of the questions about validity and certification raised by van Helmond et al , and we With regard to the interpretation of the accuracy of the absolute BP values in our home measurement study, we disagree with van Helmond et al . The cliFinally, we agree with van Helmond et al  that a mWe hope that our joint effort to scientifically examine these new devices leads to optimization of self-monitoring technology and eventually to improved patient care.


Natural product research continues to be a productive source of unusual chemistry, producing novel compounds for biomedical applications and, increasingly, sustainably providing commercially useful compounds. Despite its long history, the technologies used in natural products research are still amenable to improvements and the introduction of novel methods, materials and reagents. This special issue highlights several recent developments and improvements in the technology for natural products research.In recent years, laboratory culture of microbes has expanded the ability of natural products researchers to examine a wider variety of organisms and facilitated the isolation and identification of rare compounds. Gurusinghe et al.  comparedRhus genus. They report that not only did the method used affect the metabolic content, but that the impact of the methods was species-dependent as well.Biological material for natural products research is often collected in remote areas with limited access to sample storage or processing technology. Collection and sample processing methods can thus impact what can be identified or isolated from the material in a laboratory setting. Juárez-Aragón et al.  quantifi2 extraction to obtain commercially useful compounds from microalgae was evaluated by Molino et al. [Radix physochlainae. Their protocol was quicker and more efficient than traditional methods.Solvent extraction is another step that greatly influences the outcome of a natural products program and for this reason alternatives to traditional organic solvents have been examined. The use of supercritical COo et al.  and Mehao et al. . Both gro et al.  used ionIn order for traditional medicines and herbal extracts to be safely introduced to the market, methods for verifying the provenance, composition and quality of the material are needed. Lee et al.  present Tsang et al.  present Biological activity can be modified dramatically by stereochemistry; thus, its determination is a critical component of natural product structure elucidation. Pearce et al.  present As the papers in this special issue show, many different facets of technology for natural product research continue to be explored. The processes impacting compound identification and discovery are being probed so that they may be better controlled, and the desired outcomes obtained. Methods to enhance quality control of herbal materials and traditional medicines, and to facilitate characterization of novel metabolites are being developed. We hope these reports will spur further developments and enable natural products research to continue to expand chemical space for the benefit of humanity.


Pure (single) cultures of microorganisms and mixed microbial communities (microbiomes) have been important for centuries in providing renewable energy, clean water and food products to human society and will continue to play a crucial role to pursue the Sustainable Development Goals. To use microorganisms effectively, microbial engineered processes require adequate control. Microbial communities are shaped by manageable deterministic processes, but also by stochastic processes, which can promote unforeseeable variations and adaptations. Here, we highlight the impact of stochasticity in single culture and microbiome engineering. First, we discuss the concepts and mechanisms of stochasticity in relation to microbial ecology of single cultures and microbiomes. Second, we discuss the consequences of stochasticity in relation to process performance and human health, which are reflected in key disadvantages and important opportunities. Third, we propose a suitable decision tool to deal with stochasticity in which monitoring of stochasticity and setting the boundaries of stochasticity by regulators are central aspects. Stochasticity may give rise to some risks, such as the presence of pathogens in microbiomes. We argue here that by taking the necessary precautions and through clever monitoring and interpretation, these risks can be mitigated. In this review paper, we highlight the impact of stochasticity in single culture and microbiome engineering. Stochasticity may give rise to some risks, such as the presence of pathogens in microbiomes. We argue here that by taking the necessary precautions and through clever monitoring and interpretation, these risks can be mitigated. AxenicA closed ecosystem maintaining a single monoclonal populationDeterministicThe absence of randomness in the development of the microbial community structure and composition, which is mainly influenced by manipulatable niche‐based factorsDriftThe random fluctuation in the number of gene variations within a population over a certain period of timeDynamicsThe change in microbial community composition in function of timeImmigrationThe addition of a specific taxon to a resident community from the species pool of the metacommunityInvasionThe establishment of an immigrant taxon within a certain niche or ecosystemSpeciationGenetic divergence within clonal populations as a result of lateral gene transfer and incorporation of foreign DNA through recombination, mutational divergence and natural selectionStochasticRandom processes that influence the development of the microbial community structure and composition, which cannot be steered or controlledet al., et al., For thousands of years, humans have been relying on microbial processes to produce a plethora of food products, such as bread, beer, vinegar, cheese and other fermented products. The industrialization of our society accelerated the development of novel technologies that directly exploit microorganisms and their unique abilities. For example, the treatment of wastewater by means of activated sludge and renewable energy recovery through anaerobic digestion is both established technologies, now in existence for over a century  cultures and microbiomes will be considered. Next, the mechanisms underlying stochasticity and the consequences (issues and opportunities) for engineered systems will be examined. Finally, the concept of stochasticity will be approached from a regulatory point of view for (in)direct human applications.Fuelled by rapid developments in (meta)genomics, transcriptomics, proteomics and metabolomics techniques, greater attention has been paid to study microbial communities in their entirety and to identify the mechanisms that shape and influence microbial ecosystems of both single cultures and microbiomes. Mainly in the last decade, scientists started to acknowledge the importance of ecological stochasticity in shaping the microbial community  are more likely to become fixed when a bacterial lineage has to adapt to a novel lifestyle that reduces its long‐term population size, such as obligate symbiosis or a limited habitat range , is powered by a broad array of deterministic factors, i.e., pairwise interactions that occur between species, such as (i) syntrophy   digestive tract, imply that these are open to different stochastic processes. In that context, two features are of particular interest, which are (i) entry of outsiders in the microbial ecosystem and (ii) exit of insiders to the outside world. The potential downsides of such an open system are twofold.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Invasion of non‐desirable microorganisms could disturb process performance or compromise safety. Invasion can be defined as the establishment of an ‘immigrant’ species in a resident microbial community  engineered processes, the key advantages, especially towards microbiomes, are also twofold.et al., et al., Homeostasis is the result of a dynamic balance of interactions between microorganisms in the community and with their environment  and ‘equipped to deal’ with to disturbances (redundancy). These examples show that stochasticity should be embraced for microbiome engineering as a natural mechanism of sustaining adequate process performance.Stochasticity is part of life when dealing with both single cultures and microbiomes in biotechnological applications and process engineering. Thus, it requires a suitable framework for the regulatory authority to deal with stochasticity in an economically feasible and safe way. This is a twofold issue, as it requires (i) accurate monitoring of stochasticity and (ii) setting of boundaries on stochasticity, regarding the system in which it is considered. A proposal to solve both issues is formulated here.et al., et al., et al., To monitor stochasticity, methods to detect changes in both single and mixed genetic and phenotypic traits are needed. Single culture phenotypic changes can be monitored through flow cytometry  should be absent in microbiomes in biotechnological applications. However, given the open feature of these systems, the invasion of potentially pathogenic species cannot be avoided and requires accurate monitoring.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., A first monitoring approach concerns the ‘fingerprinting’ approach in which the degree of microbial community dynamics, i.e., the change in microbial community composition in function of time can be evaluated. Such a fingerprint and its evolution in function of time can be obtained on the genetic level through 16S rRNA gene amplicon sequencing  the initial status of the microbial community, (ii) the deterministic processes  influencing the microbial community and (iii) the (change in) input streams, e.g., the absence of pathogens and ARGs, for which the ‘omics’ or targeted methods are needed, as discussed above. Taking these three conditions into consideration, if fingerprinting methods do not indicate a deviation from the background variation, no additional analyses nor direct actions are needed. Deviations from the background dynamics profile warrant further investigation to pinpoint potential stochastic hazards and take necessary actions.et al., et al., et al., et al., et al., Even though these fingerprinting techniques provide an important overall view on the microbial community dynamics and distinguish background dynamics from strong deviations or sudden changes, they fail to detect the gradual potential influx of pathogens. Hence, identification at the lowest most precisely possible taxonomic level is mandatory to identify potential pathogens. For this, 16S rRNA gene amplicon sequencing falls short on two levels, i.e., (i) the limited depth of phylogenetic identification  can take place through basic fingerprinting methods, as mentioned earlier. The selection of the appropriate fingerprinting method depends on the system, as amplicon sequencing at the DNA level can be considered suitable for processes in which the outcome does not directly link to human consumption, such as anaerobic digestion and activated sludge systems.®) obtained by growing a single culture of Methylococcus capsulatus under semi‐sterile conditions was eventually characterized by the presence of heterotrophic bacterial strains, which fill a key functional niche necessary to maintain acceptable process performances, i.e., the degradation of the organic carbon produced by Methylococcus capsulatus  treatment, energy recovery, drinking water supply and food processing, and, therefore, cannot be eliminated from our present society. Complete elimination of all risks in microbiomes is impossible (tolerance), due to the stochastic nature of the microbiome. However, if the necessary precautions and above‐mentioned level of monitoring, e.g., on site via the MinION™ device (Maestri None declared.


Metastasis is a major cause of morbidity and mortality in cancer patients. However, the molecular and cellular mechanisms underlying the ability of cancer cells to metastasize remain relatively poorly understood. Among all solid tumors, small cell lung cancer (SCLC) has remarkable metastatic proclivity, with a majority of patients diagnosed with metastatic disease. Our understanding of SCLC metastasis has been hampered for many years by the paucity of material from primary tumors and metastases, as well as the lack of faithful pre‐clinical models. Here, we review recent advances that are helping circumvent these limitations. These advances include methods that employ circulating tumor cells from the blood of SCLC patients and the development of diverse genetically engineered mouse models of metastatic SCLC. New insights into the cellular mechanisms of SCLC metastasis include observations of cell fate changes associated with increased metastatic ability. Ongoing studies on cell migration and organ tropism promise to expand our understanding of SCLC metastasis. Ultimately, a better molecular understanding of metastatic phenotypes may be translated into new therapeutic options to limit metastatic spread and treat metastatic SCLC. Small cell lung cancer (SCLC) is highly metastatic, but rare resections of SCLC tumors and metastases have hampered our understanding of SCLC progression. This review offers a comprehensive overview of recent advances on models and mechanisms of SCLC metastasis. Circulating tumor cells (CTCs)Cancer cells that have left a tumor and circulate in the bloodstream.Epithelial‐to‐mesenchymal transition (EMT)The process by which epithelial cells acquire mesenchymal features. EMT in cancer is often associated with increased metastatic potential.Immune checkpoint inhibitorsInhibitors of negative regulators of immune reactions. Upon inhibition of these immune checkpoints, the anti‐cancer activity of immune cells can be enhanced.Macro‐metastasisA metastasis that can be detected without a microscope, often exceeding 2 mm in diameter in a cancer patient.Metastatic cascadeThe series of steps by which cancer cells leave the primary tumor and form metastases.Micro‐metastasisA small metastasis that can be best detected microscopically.Neuroendocrine tumorsTumors in which cancer cells have traits similar to those of both nerve cells and hormone‐producing cells.Pleural effusionExcess fluid around the lung. The growth of lung cancer cells outside of the lungs often generates pleural effusions.Tumor organoidsin vivo characteristics of tumors.Cellular clusters derived from tumors that mimic the three‐dimensional Vasculogenic mimicry (VM)The process by which cancer cells acquire features of vascular cells and organize themselves into blood vessel‐like structures.et al, Cancer is a leading cause of death, resulting in close to 9 million deaths worldwide every year. Tissue destructive macro‐metastases disrupt organ function and are a major source of morbidity and lethality for almost all solid tumor types  cancer types necessitates the analysis of cancer type specific molecular and cellular programs that contribute to metastasis.Cells progressing through the metastatic cascade must employ a series of diverse cellular processes, from local invasion and intravasation into the bloodstream, survival in circulation, and extravasation, to colonization and growth in new organ environments. These different steps are associated with different molecular programs that regulate migration, survival, and proliferation of cancer cells  is a highly metastatic and recalcitrant carcinoma. While worldwide data for SCLC are not available, it is estimated that SCLC accounts for ~ 15% of lung cancers and causes more than 200,000 deaths per year . A seminal study found that SCLC patients have uniquely high numbers of CTCs  and p53 (Trp53) enable the homozygous inactivation of these genes in lung epithelial cells of adult mice using adenoviral vectors expressing Cre recombinase (Ad‐Cre)  tumor suppressors in the context of the RP model. Inactivation of these genes, which are each recurrently mutated in human SCLC  shows rapid growth of SCLC tumors that are highly metastatic and metastasize within weeks of initiation  (see below), metastatic programs of the Pten or Crebbp mutant SCLC‐A subtype or the RPM SCLC‐N subtype have not yet been described. As mouse models for specific SCLC subtypes and genotypes are developed, molecular and functional characterization of the programs that drive their metastatic ability will provide further insights into how metastases differ across this heterogeneous cancer. Investigating how the different genetic alterations that drive initiation and growth affect metastatic programs will eventually help identify specific metastatic programs that can be targeted in a more precise manner.In addition to the coincident inactivation of additional tumor suppressor genes, members of the MYC family of oncogenes are frequently amplified in human SCLC  that express the neuroendocrine marker CGRP (using Adeno‐CGRP‐Cre‐mediated deletion of Rb/p53/p130), the resulting metastatic SCLC did not upregulate NFIB  were transplanted subcutaneously, they engrafted and grew tumors, but did not generate metastases. However, subcutaneous tumors generated from a mixed population of NE and non‐NE SCLC cells were uniquely capable of generating metastasis from the NE cells  is a process through which cancer cells acquire features of endothelial cells, enabling et al, Another distinct population of non‐NE SCLC cells can be generated by activation of Notch signaling in the tumor microenvironment, and this population can promote the growth of NE SCLC cells, including following chemotherapy treatment  via β1‐integrin could activate PI3K signaling and promote the survival of SCLC cells following DNA damage  has not yet been studied. Such interactions between SCLC cells could be particularly important for clusters of CTCs found in the blood of patients  is an atypical ligand for NOTCH receptors that is expressed on SCLC stem cells  is an evolutionarily conserved program that has been implicated in enhancing mobility, invasion, and resistance to cell death during metastasis (Chaffer et al, et al, et al, et al, et al, et al, et al, et al, in vivo in both mouse and human models, facilitate the migration of SCLC cells in culture in a manner similar to neuroblast migration (Yang et al, in vivo remains unknown. Knock‐down of several diverse genes associated with the formation of these protrusions in SCLC cell lines did not affect subcutaneous tumor growth, but decreased the number of liver metastases after intravenous transplantation (Yang et al, et al, It has long been recognized that SCLC cells can express neuronal markers (reviewed in (Onganer et al, et al, et al, et al, et al, et al, et al, et al, et al, Small cell lung cancer tumors commonly metastasize to the brain, liver, bones, and distant lymph nodes, but also metastasize at lower frequencies in many other organs (Froudarakis, RP model increased kidney and bone metastases (Semenova et al, et al, et al, Interestingly, the genetic alterations in SCLC may also influence the locations of metastases. Notably, overexpression of NFIB in the et al, et al, et al, et al, et al, et al, et al, et al, Brain metastases occur in around 15–20% of SCLC patients at the time of diagnosis (Nakazawa et al, et al, et al, et al, et al, RP mouse model with overexpression of NFIB (Semenova et al, Another common site for SCLC metastasis is the bone, with around 20% of SCLC patients developing bone metastases (Nakazawa et al, Small cell lung cancer is an aggressively metastatic cancer. While an increase in knowledge of the genetic drivers of lung adenocarcinoma has enabled the development of new treatments that have extended survival and decreased mortality, the decrease in mortality from SCLC has been strictly driven by decreased incidence (Howlader et al, et al, et al, et al, et al, Differences in treatment responses between different models of SCLC are becoming increasingly apparent (Ferone in vitro phenotypes associated with metastasis, in vivo metastases that form from xenograft transplants, and metastases that form in genetically engineered mouse models. While these models are informative, they each have limitations. For example, models of metastatic SCLC based on cell lines can potentially reflect phenotypes gained during propagation in culture, and existing genetically engineered mouse models do not yet reflect the full range and frequency of metastases in patients. While promising new models like CDXs address some of these limitations, including the development of brain metastases in some CDX models, there are still important gaps in our ability to model key aspects of SCLC metastasis. For example, studying SCLC metastases in xenograft and autochthonous mouse models is limited to metastatic tumors that develop before the mice succumb to their primary tumors. New models that overcome or circumvent these differences in frequency of metastases in specific organs will be important to address additional aspects of organ tropism of SCLC. For example, irradiating primary lung tumors at late stages in autochthonous mouse models, generating primary tumors in only one lobe followed by partial pneumonotomy, or surgically removal of subcutaneous xenografts before they grow too large may provide more time for metastases to grow in other organs.The development of additional models of SCLC remains critical to further deconvolute the cellular and molecular mechanisms that drive SCLC metastasis. Current experimental systems rely on et al, et al, et al, et al, et al, et al, Furthermore, many new therapies for SCLC are focused on the interactions between cancer cells and immune cells. Both adaptive and innate immune cells can play significant roles at different stages of cancer growth and the metastatic process. In other cancer types, immune responses have pro‐ and anti‐tumorigenic effects on primary tumor growth, influence dissemination, and regulate metastatic seeding and growth (Kitamura Despite decades of being understudied, SCLC may soon be leading our conceptual understanding of the determinants of metastatic ability. With a growing understanding of the impact of the cell type of origin and cancer genetics, we are poised to uncover the spectrum of cellular and molecular mechanisms that influence SCLC metastasis. The certain complexity of these mechanisms can now be embraced rather than viewed as insurmountable task. Understanding the cellular and molecular drivers of each step of SCLC development remains a formidable undertaking. While the identification of genetic and cellular heterogeneity adds multiple layers of complexity to this disease, new models are rapid enhancing our ability to study each cellular process in the metastatic cascade and connect molecular mechanisms to cellular phenotypes. Identification of cellular and molecular mechanisms that drive metastatic potential will ultimately provide targets for the development of therapies to block the development of metastatic SCLC, prevent the growth of additional metastatic tumors during and after treatment, and treat multi‐organ metastatic disease.J.S. receives research funding from Stemcentrx/Abbvie and Pfizer, and licensed a patent to Forty Seven Inc./Gilead on the use of CD47 blocking strategies in SCLC. M.M.W. has equity in, and is an advisor for, D2G Oncology Inc.https://www.cancer.org/cancer/lung‐cancer/if‐you‐have‐small‐cell‐lung‐cancer‐sclc.htmlA lay person description of small cell lung cancer and treatment options by the American Cancer Society.https://www.omim.org/entry/182280A resource page on SCLC from the Online Mendelian Inheritance in Man online catalog of human genes and genetic disorders.https://www.metastasis‐research.org/An international professional society that supports research on processes fundamental to metastasis in various cancer types.https://depmap.org/portal/A database that includes a number of human SCLC cell lines and the consequences of gene knock‐down or knock‐out.https://www.cbioportal.org/A portal for cancer genomics that includes human SCLC tumors.Develop new pre‐clinical models that faithfully recapitulate metastasis in SCLC patients, including organ tropism.Gain a better understanding of how the cell type of origin, specific genomic alterations, and cell–cell interactions influence metastatic development and response to therapy.Identify therapeutic strategies that can inhibit metastatic spread in early‐stage SCLC patients and that can block metastatic growth in late‐stage SCLC patients.


As reports from McKinsey Global Institute . Therefore, there is an urgent need to protect machine learning models against potential attacks. Although there is an active research directions for addressing adversarial attacks in machine learning (e.g., Zhou et al., All 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.


The conditions, timing, and setting for the origin of life on Earth and whether life exists elsewhere in our solar system and beyond represent some of the most fundamental scientific questions of our time. Although the bombardment of planets and satellites by asteroids and comets has long been viewed as a destructive process that would have presented a barrier to the emergence of life and frustrated or extinguished life, we provide a comprehensive synthesis of data and observations on the beneficial role of impacts in a wide range of prebiotic and biological processes. In the context of previously proposed environments for the origin of life on Earth, we discuss how meteorite impacts can generate both subaerial and submarine hydrothermal vents, abundant hydrothermal–sedimentary settings, and impact analogues for volcanic pumice rafts and splash pools. Impact events can also deliver and/or generate many of the necessary chemical ingredients for life and catalytic substrates such as clays as well. The role that impact cratering plays in fracturing planetary crusts and its effects on deep subsurface habitats for life are also discussed. In summary, we propose that meteorite impact events are a fundamental geobiological process in planetary evolution that played an important role in the origin of life on Earth. We conclude with the recommendation that impact craters should be considered prime sites in the search for evidence of past life on Mars. Furthermore, unlike other geological processes such as volcanism or plate tectonics, impact cratering is ubiquitous on planetary bodies throughout the Universe and is independent of size, composition, and distance from the host star. Impact events thus provide a mechanism with the potential to generate habitable planets, moons, and asteroids throughout the Solar System and beyond.  But how did life on Earth begin? There is some consensus regarding the requisite conditions for the transition from prebiotic chemistry to living systems: a maintained excess of Gibbs free energy (-ΔG), a solvent (water), a mode for the encapsulation and concentration of prebiotic molecules, a mechanism of information storage, and the presence of catalytic molecules such as enzymes , which 13C values within carbonaceous inclusions found in apatite grains , and synthesized with results from other studies. We review the beneficial effects for microbial life ranging from generating conditions conducive to the origin of life  to varied habitats for life that persist long after an impact event, including transient hydrothermal systems, endolithic habitats in impact glasses and impact shocked rocks, and impact crater lakes. The purpose of this contribution is not to repudiate or argue against any previously proposed environments for the origin of life, but to synthesize ideas and data on the potential importance of meteorite impact craters. While impact craters represent a standalone environment, we show that they can also provide an alternative mechanism or pathway by which to generate previously proposed prebiotic environments, such as submarine hydrothermal vents, subaerial hydrothermal fields, and even pumice rafts. Furthermore, unlike other geological processes such as volcanism or plate tectonics, impact cratering is ubiquitous on rocky and icy bodies in the Solar System , and the high strain rates involved (∼104 s−1 to 106 s−1) set the impact cratering process apart from any other geological process, such as volcanism and tectonism . From a [et al. : (1) con [et al.  suggesteet al. , there is a notable increase in porosity and permeability in target rocks , the transient cavity undergoes only minor modification, which results in the formation of a bowl-shaped simple crater  lined wiet al.,i.e., for most objects in the Universe), impact cratering will thus be the primary agent of resurfacing, and impact ejecta deposits and central uplifts will be the primary mechanism to exhume ultramafic rocks, such as peridotite and basalt, from the deep subsurface. Furthermore, it is thought that plate tectonic rates during the Hadean were slower than the present-day and that the Hadean oceanic crust was much thicker  of the original transient cavity and to an even smaller proportion of the final crater. Indeed, it has been shown that even within rocks that were highly shocked and encased in impact melt rocks, bioessential elements  involved. The three dominant groups of alteration minerals in terrestrial craters are clays, zeolites, and carbonates  of target rock and fluid sources  and habitats (see Section 4) necessary to support the origins and early evolution of life.et al.,et al.,et al.,et al.,Once the impact-generated heat has fully dissipated from a crater and hydrothermal circulation has ceased, ambient surface temperatures return to pre-impact values: we consider this as the beginning of the next and longest-lived phase of post-impact ecological succession  that wilet al.,et al.,et al.,et al. . On Earth, this process is significant, and it is primarily driven by plate tectonics and wind/hydrological erosion. Although atmospheric density leads to differences in impact dynamics on Earth and Mars, the characterization of 198 craters on Earth . Meteorites can be classified into three main groups  but new biologically relevant molecules, including threose, erythrose, and ethylene glycol, were formed.In an intriguing set of experiments, McCaffrey et al.  mixed thA synthetic view of the conditions at the site of impact craters leads to a number of conditions that could be conducive to an origin of life. Cockell  suggesteet al.  crae.g., Newsom, i.e., vugs and veins) to being completely pervasive. The difference in intensity of hydrothermal alteration of crater-fill impactites has been ascribed to the presence/absence of an overlying crater lake . The authors outlined a series of metabolic pathways whereby chemolithotrophic microorganisms could have been involved in the weathering of hydrothermal sulfides. Sulfides are ubiquitous in impact-generated hydrothermal systems , sulfid [et al. , charact Naumov,  and provet al.,et al.,et al.,et al.,et al.,et al.,Is there evidence that these impact-generated hydrothermal systems were colonized by microorganisms? The most conclusive evidence thus far is sulfur isotope data from the Haughton . Rapid cooling or “quenching” of silicate impact melt results in the production of glass  and often containing quench crystallites of minerals such as pyroxene and feldspar  for liquid water to exist. However, as discussed by Neish et al. , transiee.g., Gold, et al.,et al.,et al.,et al.,et al.,et al.,In recent years, there has been a growing recognition of the importance of the deep subsurface biosphere on Earth  than New Quebec likely owing to the increased area for littoral zone organisms , complexification , and plausibility . Westall et al.  in the acidic Hadean ocean, satisfying the requirement for a -ΔG. Furthermore, the resulting pH gradient between the highly alkaline effluent and the acidic ocean could have driven the first primitive proton pumps across a hydrothermal mound. These initial processes would have allowed for increased complexity and the generation of pyrophosphate in ferrous hydroxide layers, acting as highly reactive and permeable “membranes,” allowing for concentration of prebiotic constituents. Although these hydrothermal vent and hydrothermal-sedimentary environments are set in the context of submarine volcanism, we contend that these processes could also have occurred in impact-generated hydrothermal environments on Hadean Earth.In Section 3, we reviewed how meteorite impacts could have delivered and generated the necessary ingredients for life—ranging from amino acids, HCN and CO, to clay minerals—on Hadean Earth. In Section 4, we discussed how impact events can also create unique new habitats for life. In this section, we return to the question of the environment (or environments) in which life originated on Earth. We draw extensively from the work of Westall et al. , who pro  but for the primary potential impact-generated habitats and substrates that they could contain.e.g., gullies, subglacial environments). Exactly when the transition from a potentially habitable surface to an inhospitable one occurred is not known. However, in extreme locations on Earth, surface conditions render the interior and subsurface of rocks, and soils beneath them, as preferential refugia for the propagation of life. Regardless of either an early warm and wet or cold and dry Mars, by the time the earliest evidence for life on Earth was recorded in the rock record, due to loss of the magnetic field by ∼3.9–4.1 Ga . In many subsurface environments, life is generally not limited by space but by energy and nutrients , to exoplanets, we suggest that the concept of habitable zones and what factors influence planetary habitability should be reconsidered with meteorite impacts in mind.Looking beyond Earth, as we have discussed in this review, we suggest that impact craters should be considered as prime sites in the search for evidence of past life on Mars and may have increased the habitability of the subsurface, with implications for the potential of extant life on Mars. While not within the scope of this review, we contend that impacts could increase the potential for any planetary body with the necessary ingredients for life—which could also be impact-delivered—to be at least transiently habitable. From large asteroids, such as Ceres, to satellites of the outer Solar System  where life originated on Earth. This is a hypothesis that could eventually be tested on Mars or other planetary bodies. Would it not be poetic that impacts, long seen as harbingers of death, turn out to have in fact been the cradle of life?


We read with interest the article by Hung et al. “Long-Term Outcomes of Sentinel Lymph Node Biopsy for Ductal Carcinoma in Situ” . The autWe do not think that this conclusion can be drawn based on the data they had available and selected for their analysis. As stated by Hung et al. , they usGuidelines on the use of SLNB in patients with biopsy-proven DCIS are divergent and so is the interpretation of these guidelines, because there also is considerable variation in the actual use of SLNB in these patients ,6. To imDisclosures: The authors have no disclosures to declare.Not applicable.


Dedicated to the memory of Arren Bar‐Even (1980‐2020), one of the most brilliant biochemists of our times.et al., et al., Living beings are the result of a cocktail made with unknown doses of chance and necessity. Consider a thought experiment, in which we could rewind the ‘tape of life’ starting from the same initial conditions, what biochemical traits and cellular features would finally be the same as those we observe today? It is clear that what is real in biology is a subset of what is possible, and this issue has been discussed at different scales. Thus, structural and dynamic developmental constraints limit the space of solutions for animal bodies Alberch, , whereasEscherichia coli K12  and well‐defined physiological processes . Describing chemical transformations in cell‐free systems and the use of inhibitors, mutants or isotope labelling helped to draw the first metabolic pathways Morange, . More reet al., 2. Experiments in prebiotic systems chemistry, exploring the origin of glycolytic intermediates, indicate an early emergence of this historically frozen transformation of C3 in C2 moieties  is an artificial pathway composed of natural enzymes from different phylogenetic origins and showing a complete carbon conservation without decarboxylation steps during the transformation of glucose in acetate (i.e. one mole of hexose delivers three moles of acetate without carbon loss). NOG functions in vitro and in vivo in E. coli, by the action of canonical glycolytic and the pentose phosphate pathway enzymes from the host in combination of an enzyme of the bifid shunt, namely a phosphoketolase from Bifidobacterium adolescentis, allowing a new rearrangement and recycling of carbon skeletons (Bogorad et al., et al. (2 emissions by cellular factories. There is still a long way from all those laboratory successes to their efficient industrial implementations.Motivated by the Nobel prize winner Frances H. Arnold, I would say that metabolism is amazing not only because what it does now, but for what it , et al. . The lonin vitro combinations of purified natural enzymes performing unnatural chemical processes. What is now certainly innovative are the approaches, the tools and the engineering ideology under current synthetic biology projects. Thus, Fessner (et al., et al., et al., et al., et al., The idea of designing new metabolic processes and catalysts adopting a non‐conformist attitude – going beyond the naturally evolved pathways and enzymes – is not completely new. For instance, Fessner and Walter  proposed Fessner  has intr


The past decades have witnessed tremendous progress in the detailed structural knowledge of proteins as potential or validated drug targets and the discovery of new drugs based on this wealth of knowledge progressed in parallel.In this Special Issue, several papers deal with the structure-based investigation of targets useful for the discovery of novel anticancer drugs. Al-Warhi et al.  targetedTargets involved in both cancer and inflammatory diseases have been the focus of intense structure-based design campaigns and are well-represented in this Special Issue. Londhe et al.  analyzed3 receptor (H3R) ligand, bearing an isothiocyanate warhead, that behaved as an inverse agonist of this G protein-coupled receptor (GPCR). Scarpino et al. [Two papers of this Special Issue deal with covalent ligands and the challenges posed by in silico analysis of covalent inhibitors. Wagner et al.  designedo et al.  designedBased on the concept that the knowledge of interactions at either a molecular or high topological level are important for the design of effective ligands, Bojarska et al.  designedMycobacterium tuberculosis survival and virulence, Battah et al. [The design of anti-infective compounds and of pest control agents is another field in which structure-based design plays a critical role for both the discovery of new agents and for the control of drug-resistance issues. Tian et al.  analyzedh et al.  identifih et al.  provided


Intracerebral hemorrhage (ICH) is a destructive form of stroke that often results in death or disability. However, the survivors usually experience sequelae of neurological impairments and psychiatric disorders, which affect their daily functionality and working capacity. The recent MISTIE III and STICH II trials have confirmed that early surgical clearance of hematomas does not improve the prognosis of survivors of ICH, so it is vital to find the intervention target of secondary brain injury (SBI) after ICH. Mitochondrial dysfunction, which may be induced by oxidative stress, neuroinflammation, and autophagy, among others, is considered to be a novel pathological mechanism of ICH. Moreover, mitochondria play an important role in promoting neuronal survival and improving neurological function after a hemorrhagic stroke. This review summarizes the mitochondrial mechanism involved in cell death, reactive oxygen species (ROS) production, inflammatory activation, blood–brain barrier (BBB) disruption, and brain edema underlying ICH. We emphasize the potential of mitochondrial protection as a potential therapeutic target for SBI after stroke and provide valuable insight into clinical strategies. Hemorrhagic stroke, which is less common but far more likely to be fatal, makes up about 13% of stroke cases. Indeed, two-thirds of stroke survivors will experience moderate or severe disability  are the causes of SBI after ICH  oxidase and myeloperoxidase  is a dynamic interface between the peripheral circulation and the CNS that prevents toxic substances from the CNS and contributes to the maintenance of brain homeostasis  and intimal anion channels (IMACs), and activation of these channels leads to an imbalance of REDOX homeostasis in cells and mitochondria, inducing ROS release  and apoptosis induction factor (AIF), which play a central role in the survival or death of neurons , a membrane protein, which is recruited into the mitochondrial outer membrane and triggers mitochondrial division (Montessuit et al., Previous studies have shown that the Drp1 inhibitor mdivi-1 can attenuate oxidative stress and neuronal apoptosis after subarachnoid hemorrhage (Fan et al., Mitophagy was first described by Lemasters , who detIn various pathological environments, “alternative” activation pathways of mitophagy are also involved in mitophagy (Ma et al., In general, it appears that the effect of mitophagy depends on its severity. Under physiological conditions, mitophagy prevents accelerated cellular senescence and programmed cell death Kang, . HoweverIn cells, the MMP is typically maintained between 80 and 140 mV (Cloonan et al., in vivo and in vitro, and some researchers have found that melatonin can significantly inhibit apoptosis and mitochondrial damage in vivo and in vitro (Wang et al., via the inhibition of mitochondrial depolarization (Zheng et al., The mitochondrial injury induced by ICH is the result of mPTP opening, whereby various proteins, such as CytoC, are released into the cytoplasm, constituting important events that lead to apoptosis (Tomasello et al., in vivo and in vitro (Li et al., In addition, Miro1 is a glutamate receptor-dependent calcium sensor that targets the mitochondria of neurons, connecting mitochondria with motor proteins (Macaskill et al., Hayakawa et al.  demonstrin vitro ischemia/reperfusion model (Han et al., Astrocytic release of extracellular mitochondria particles was mediated by a calcium-dependent mechanism involving CD38/cyclic ADP ribose signaling. Transient focal cerebral ischemia in mice induced astrocytic mitochondria entry to adjacent neurons that amplified cell survival signals (Hayakawa et al., Studies have shown that the main function of transferred mitochondria is to enhance the capacity of recipient cells to metabolize energy (Picca et al., Mitochondrial dysfunction is an early initiating event in the pathophysiology of ICH. Bioenergy genetic defects, structural abnormalities and mitochondrial morphology, and aberrant mitochondrial dynamics play an important role in the activation of neuronal death signaling pathways. In preclinical studies, interventions aimed at mitochondrial quality control and mitochondrial dynamics have been shown to have neuroprotective effects (Chimeh et al., Recent evidence of mitochondrial transfer has provided a new perspective for intercellular communication. The latest research suggests that mitochondria themselves can act as “help me” signals to respond to different extracellular stimuli and recruit adjacent cells to rescue those injured in stroke and those affected by aging, and disease (Heyck et al., Here, The clinical trials using mitochondrial protectants or antioxidants have not been successful or fulfilled yet. ROS and mitochondrial damage are produced rapidly and have a cascade amplification effect (Qu et al., WC, CG, and YC drafted the manuscript and figures. HF and YC proofread and revised the manuscript. YC gave the final proof for this submission. All authors contributed to the article and approved the submitted version.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.


Dear Editor,With great interest we have read the recent systematic review on “Nonspecific Complaints (NSCs) in the Emergency Department”  and woulHowever, NSCs were first defined by Nemec et al  This oriSurprisingly, the original publication by Nemec et al. , coiningTherefore, we suggest that further research should be based on a common definition of nonspecific complaints in emergency presentations, in order to reduce heterogeneity of studies.


Cancer cachexia has been recognized as a major, life‐limiting complication in the treatment of cancer patients and is composed of distinctive stages: pre‐cachexia, cachexia, and refractory cachexia.Journal of Cachexia, Sarcopenia and Muscle, Morigny et al. have addressed the alterations in bioactive lipids associated with cancer cachexia in pre‐clinical mouse models  and complemented the results with the analysis of cancer patients with or without cachexia. The lipidome analysis performed by the authors included 1100 lipid species, and the results show that several bioactive lipids were regulated in cachexia. Sphingolipids (for comprehensive description of the sphingolipid metabolism, see Gault et al.et al.In the current issue of the The authors have no conflict of interest.


Understanding temperature‐induced variations in body size is therefore of fundamental and applied interest, yet thermal responses of body size remain poorly understood. Temperature–size (T–S) responses tend to be negative , which has been termed the temperature–size rule (TSR). Explanations emphasize either physiological mechanisms  or the adaptive value of either a large body size (e.g. to increase fecundity) or a short development time . Oxygen limitation could act as a proximate factor, but we suggest it more likely constitutes a selective pressure to reduce body size in the warm: risks of oxygen limitation will be reduced as a consequence of evolution eliminating genotypes more prone to oxygen limitation. Thus, T–S responses can be explained by the ‘Ghost of Oxygen‐limitation Past’, whereby the resulting (evolved) T–S responses safeguard sufficient oxygen provisioning under warmer conditions, reflecting the balance between oxygen supply and demands experienced by ancestors. T–S responses vary considerably across species, but some of this variation is predictable. Body‐size reductions with warming are stronger in aquatic taxa than in terrestrial taxa. We discuss whether larger aquatic taxa may especially face greater risks of oxygen limitation as they grow, which may be manifested at the cellular level, the level of the gills and the whole‐organism level. In contrast to aquatic species, terrestrial ectotherms may be less prone to oxygen limitation and prioritize early maturity over large size, likely because overwintering is more challenging, with concomitant stronger end‐of season time constraints. Mechanisms related to time constraints and oxygen limitation are not mutually exclusive explanations for the TSR. Rather, these and other mechanisms may operate in tandem. But their relative importance may vary depending on the ecology and physiology of the species in question, explaining not only the general tendency of negative T–S responses but also variation in T–S responses among animals differing in mode of respiration (e.g. water breathers  I.Body size is central to ecology at multiple scales, from organismal fecundity to the functioning of communities and ecosystems , that is they mature at a smaller size when reared in warmer conditions, despite initially growing faster , has in itself been used to argue that the TSR is adaptive , when reared under warmer conditions. Size‐at‐stage results from the interplay between the rate of growth and the length of the period spent growing, and therefore a faster growth to a smaller size in the warm (i.e. the TSR) arises logically from warming stimulating development rate more than growth rate. Body size responses to temperature vary both in strength and sign (i.e. increases or decreases) across species. Consequently, the field has moved to a more quantitative approach examining the magnitude and direction of size responses to temperature et al. , we will also describe the substantial variation in responses across different taxa and environments . Taking a meta‐analytical perspective, Horne et al.  extended et al. et al., et al., Differences in thermal sensitivity of growth and development rates give rise to T–S responses et al., et al., Debates continue on whether or not growth rate is increasingly constrained during ontogeny, and if so, whether or not warmer temperature increases these constraints, leading to smaller size at maturity or final size  increase surface area for resource uptake during growth, such as by changing body shape  increase rates at which they obtain and distribute resources  reduce rates of demand for resources . These adjustments to the rate of resource uptake and demand reduce the likelihood that the constraints will be observed directly in controlled laboratory studies of the TSR, which provide abundant food, levels of oxygen availability typical for the species, non‐extreme  temperatures and an absence of predators and disease. Even in the field, such constraints may be observed only occasionally. On the other hand, overcoming or avoiding these physical constraints is unlikely to be cost‐free. Thus, organisms may have adapted so that they are not ‘panting for breath’ during normal growth, but could nonetheless experience resource limitation under more demanding conditions (e.g. when pursued by predators or encountering pathogens). Although such demanding conditions may be rare, they are also disproportionately detrimental. Organisms should therefore maintain a safety margin (e.g. aerobic scope) to prevent resource limitation of growth and reduction in fitness, exemplified by the maintain aerobic scope and regulate oxygen supply (MASROS) model, in which size is adjusted to maintain sufficient oxygen supply relative to demand et al., et al., et al., et al., et al., et al., et al., In order to grow, organisms need resources such as food and oxygen, which together shape the energy budget of an organism. Changes in the energy budgets and energy allocation with temperature have been used to explain the TSR  was highest in the youngest instars which lack air sacs and rely more on diffusive gas exchange  for metabolism to be independent of adult body size across a range of insect species. Larger species likely prevent progressive oxygen limitation with increasing body size by having greatly increased tracheal dimensions and these do appear to set upper limits to the size that insects may attain et al., et al., et al., et al., et al., et al., Fish have been documented to adhere to the TSR Manduca sexta, the decision to moult or pupate is size and oxygen dependent. As animals increase their body mass, their demand for oxygen also increases, but since the tracheal system can only be enlarged upon moulting they cannot correspondingly increase the capacity for oxygen supply. Such low capacity for oxygen supply relative to demand triggers the endocrine cascade that advances development  developed into smaller adults when reared under hypoxia, but more than doubled the number of moults to get there compared to normoxia. Under hyperoxia there were fewer moults, supporting the idea that oxygen availability directly influences developmental processes.Effects of oxygen and food are not limited to growth rate, but may also act on development Table . Callieret al., et al., et al., et al., et al., via basking. This could select for a reduced development rate, or a lower thermal sensitivity of development which avoids leaving insufficient time for completing growth in terms of mass, thus explaining converse TSR in heliotherms A notable exception to the near‐universal pattern of size reductions with warming are the grasshoppers. Grasshoppers could be less inclined to follow the TSR for several reasons.First, oxygen may be less limiting in larger terrestrial arthropods such as grasshoppers, because gas exchange in their tracheal network relies more on convection et al., et al., via changing cell size  resulted in a 50% increase in cell size, resembling the 50% increase in genome size  and mTOR  regulatory pathways  of 4 pg or 3.91 × 109 base pairs], and the weakest T–S responses were observed in the species with the smallest genome (T. crassus: ~ 0.8 pg). Horne et al.  risks of oxygen limitation are more likely to arise in tissues made up of large cells, animals with larger cells may be more likely to reduce cell size plastically to improve oxygen provisioning. If variation in cell number is small, such changes in cell size will be reflected in stronger T–S responses in body size. Triploid tadpoles of the frog , et al.  reportede et al.  report det al., et al., et al., et al., Genome size is also linked to development rate, with large genomes being associated with slower development in fruit flies  were reared for multiple generations, the smallest size was observed in fish reared under temperatures that were warmer than they had previously experienced ad libitum, growth rates could be adaptively modulated as a result of warming exacerbating resource limitation during the species’ evolutionary history  the TSR, while the TSR is strengthened if development rates increase with temperature more than do growth rates. We have seen why warming‐induced acceleration is more likely curtailed for growth: although limited oxygen or food is unlikely to constrain growth rates directly when provided V.et al., vice versa via increased competition, predation or resource scarcity (food or oxygen). Such increased mortality risks may be associated with thermal acceleration of physiological rates , whose resultant energetic costs are known to impair immune function per capita resource demand and both their aerobic scope and their ability to obtain sufficient food or oxygen may be less capable of matching warming‐enhanced demand compared with that of smaller individuals Lecane inermis, gains in fecundity with body size were dependent not only on temperature, but also on oxygen levels  populations, the reproductive advantage of larger body size decreased with increasing site temperature within 100 years sensu Blanckenhorn & Demont, Lycaena hippothoe than in multivoltine populations. Similarly, perceived time available can alter T–S responses (see Section Lestes sponsa (i.e. a converse James’ cline). Grasshoppers have a diapause at the egg stage, enforcing univoltism, which may explain why grasshoppers generally exhibit converse T–S responses . The benefits of early breeding or even completion of an extra generation may be substantial Cole, , and canet al., et al. et al., et al., et al., et al., et al., et al., et al., i) the voltinism of the population from which the individuals originated; (ii) the physiological state of the individual; and (iii) the temperature and light regime of its environment  et al., et al., et al., et al., et al., et al., et al., The proximate mechanisms by which a shortage of oxygen puts large individuals in the warm at a disadvantage, especially in aquatic environments (see Section (4)via behavioural thermoregulation, reducing the information content of air temperature. Hypoxia is also more common in aquatic environments, especially during warm periods. The ‘Ghost of Oxygen‐limitation Past’ is therefore more likely to operate in aquatic ectotherms, favouring genotypes that grow to a smaller size in warm water, and consequently avoiding oxygen constraints on growth. This prediction is consistent with the stronger TSR in aquatic than in terrestrial species  and food conditions? How do T–S responses affect community assembly by affecting geographic range shifts, predator–prey relationships and phenology?Extend the scope of data on T–S responses. How does developmental temperature affect body size in larger‐bodied species, both aquatic and terrestrial? How does size in aquatic species change across altitudinal clines? How do T–S responses change over multiple generations when long‐term adaptation also starts to play a role? Do multiple‐generation T–S responses differ if temperature is decoupled from proposed selective factors ?Create a database for T–S responses, facilitating future (meta) analyses.Explain the adaptive nature of T–S responses by integrating growth trajectories of juveniles to fitness consequences of adults  are decoupled from temperature. Are larger individuals more susceptible to oxygen limitation under warmer conditions and does such (incipient) oxygen limitation proximately or ultimately limit growth?We are beginning to understand the distinct patterns in T–S responses across animal groups. Still, many important questions remain, and here we propose some questions and directions for future research:VIII.The many explanations proposed Table  for obseEffects of temperature are pervasive, affecting biological levels of organization ranging from whole‐organism growth performance down to activities of individual proteins, which makes it unlikely that a single proximate mechanism underlies the TSR. An oxygen perspective may help to explain the effects of temperature on size, especially in large aquatic ectotherms, which are arguably most susceptible to risks of oxygen limitation.Warming may exacerbate risks of oxygen limitation or reduce the safety margin of aerobic scope. For air breathers such as terrestrial insects, problems with insufficient oxygen may be less likely and time constraints take centre stage. Season length may constrain developmental period, forcing them to prioritize time over size as overwintering is more challenging. Day length and temperature may together provide information on how long conditions will remain favourable for development, explaining why thermal responses are modulated by photoperiod.via oxygen provisioning) and for safeguarding completion of development (via time sensing). As such, oxygen supply can be both a proximate mechanism and an ultimate driver (the ‘Ghost of Oxygen‐limitation Past’).Time constraints, mortality risks, and resource limitation are not mutually exclusive explanations for the TSR. Rather, they may operate in tandem but their relative importance may vary depending on the ecology and physiology of the species in question Fig. . At the