From coarse to fine: the absolute Escherichia coli proteome under diverse growth conditions

Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E. coli.     

A multi‐year dormancy strategy in a cabbage beetle population in southeastern China

The life cycle of the cabbage beetle Colaphellus bowringi in southeastern China is complex due to four options for adult development: summer diapause, winter diapause, prolonged diapsuse, and nondiapause. However, detailed information on the multi‐year emergence patterns of diapausing individuals in this beetle has not been documented. In this study, we monitored the adult emergence patterns of diapausing individuals and estimated the influence of the diapause‐inducing temperature and photoperiod on the incidence of prolonged diapause under seminatural conditions for several years. The duration of diapause for adults collected from the vegetable fields in different years varied from several months to 5 years. Approximately 25.9%–29.2% of individuals showed prolonged diapause (emergence more than 1 year after entering diapause) over the 5 years of observation. Furthermore, regardless of insect age, the emergence of diapausing adults from the soil always occurred between mid‐February and March in spring and between late August and mid‐October in autumn, when the host plants were available. The influence of diapause‐inducing temperatures (22, 25, and 28°C) combined with different photoperiods (L:D 12:12 h and L:D 14:10 h) on diapause duration was tested under seminatural conditions. Pairwise comparisons of diapause duration performed by the log‐rank test revealed that the low temperature of 22°C combined with the long photoperiod of L:D 14:10 h induced the longest diapause duration, whereas the low temperature of 22°C combined with the short photoperiod of L:D 12:12 h induced the highest proportion of prolonged diapause. This study indicates that C. bowringi adopts a multi‐year dormancy strategy to survive local environmental conditions and unpredictable risks.     

Co‐occurrence models fail to infer underlying patterns of avoidance and aggregation when closure is violated

Advances in multi‐species monitoring have prompted an increase in the use of multi‐species occupancy analyses to assess patterns of co‐occurrence among species, even when data were collected at scales likely violating the assumption that sites were closed to changes in the occupancy state for the target species. Violating the closure assumption may lead to erroneous conclusions related to patterns of co‐occurrence among species. Occurrence for two hypothetical species was simulated under patterns of avoidance, aggregation, or independence, when the closure assumption was either met or not. Simulated populations were sampled at two levels (N = 250 or 100 sites) and two scales of temporal resolution for surveys. Sample data were analyzed with conditional two‐species occupancy models, and performance was assessed based on the proportion of simulations recovering the true pattern of co‐occurrence. Estimates of occupancy were unbiased when closure was met, but biased when closure violations occurred; bias increased when sample size was small and encounter histories were collapsed to a large‐scale temporal resolution. When closure was met and patterns of avoidance and aggregation were simulated, conditional two‐species models tended to correctly find support for non‐independence, and estimated species interaction factors (SIF) aligned with predicted values. By contrast, when closure was violated, models tended to incorrectly infer a pattern of independence and power to detect simulated patterns of avoidance or aggregation that decreased with smaller sample size. Results suggest that when the closure assumption is violated, co‐occurrence models often fail to detect underlying patterns of avoidance or aggregation, and incorrectly identify a pattern of independence among species, which could have negative consequences for our understanding of species interactions and conservation efforts. Thus, when closure is violated, inferred patterns of independence from multi‐species occupancy should be interpreted cautiously, and evidence of avoidance or aggregation is likely a conservative estimate of true pattern or interaction.     

Biological and molecular profile of fracture non‐union tissue: A systematic review and an update on current insights

Fracture non‐union represents a common complication, seen in 5%–10% of all acute fractures. Despite the enhancement in scientific understanding and treatment methods, rates of fracture non‐union remain largely unchanged over the years. This systematic review investigates the biological, molecular and genetic profiles of both (i) non‐union tissue and (ii) non–union‐related tissues, and the genetic predisposition to fracture non‐union. This is crucially important as it could facilitate earlier identification and targeted treatment of high‐risk patients, along with improving our understanding on pathophysiology of fracture non‐union. Since this is an update on our previous systematic review, we searched the literature indexed in PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library using Medical Subject Heading (MeSH) or Title/Abstract words (non‐union(s), non‐union(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs) from August 2014 (date of our previous publication) to 2 October 2021 for non‐union tissue studies, whereas no date restrictions imposed on non–union‐related tissue studies. Inclusion criteria of this systematic review are human studies investigating the characteristics and properties of non‐union tissue and non–union‐related tissues, available in full‐text English language. Limitations of this systematic review are exclusion of animal studies, the heterogeneity in the definition of non‐union and timing of tissue harvest seen in the included studies, and the search term MSC which may result in the exclusion of studies using historical terms such as ‘osteoprogenitors’ and ‘skeletal stem cells’. A total of 24 studies (non‐union tissue: n = 10; non–union‐related tissues: n = 14) met the inclusion criteria. Soft tissue interposition, bony sclerosis of fracture ends and complete obliteration of medullary canal are commonest macroscopic appearances of non‐unions. Non‐union tissue colour and surrounding fluid are two important characteristics that could be used clinically to distinguish between septic and aseptic non‐unions. Atrophic non‐unions had a predominance of endochondral bone formation and lower cellular density, when compared against hypertrophic non‐unions. Vascular tissues were present in both atrophic and hypertrophic non‐unions, with no difference in vessel density between the two. Studies have found non‐union tissue to contain biologically active MSCs with potential for osteoblastic, chondrogenic and adipogenic differentiation. Proliferative capacity of non‐union tissue MSCs was comparable to that of bone marrow MSCs. Rates of cell senescence of non‐union tissue remain inconclusive and require further investigation. There was a lower BMP expression in non‐union site and absent in the extracellular matrix, with no difference observed between atrophic and hypertrophic non‐unions. The reduced BMP‐7 gene expression and elevated levels of its inhibitors (Chordin, Noggin and Gremlin) could potentially explain impaired bone healing observed in non‐union MSCs. Expression of Dkk‐1 in osteogenic medium was higher in non‐union MSCs. Numerous genetic polymorphisms associated with fracture non‐union have been identified, with some involving the BMP and MMP pathways. Further research is required on determining the sensitivity and specificity of molecular and genetic profiling of relevant tissues as a potential screening biomarker for fracture non‐unions.     

Variety of size and form of GRM2 bacterial microcompartment particles

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi‐permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non‐native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo‐EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi‐layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.     

Intraseasonal predictability of natural phytoplankton population dynamics

It is difficult to make skillful predictions about the future dynamics of marine phytoplankton populations. Here, we use a 22‐year time series of monthly average abundances for 198 phytoplankton taxa from Station L4 in the Western English Channel (1992–2014) to test whether and how aggregating phytoplankton into multi‐species assemblages can improve predictability of their temporal dynamics. Using a non‐parametric framework to assess predictability, we demonstrate that the prediction skill is significantly affected by how species data are grouped into assemblages, the presence of noise, and stochastic behavior within species. Overall, we find that predictability one month into the future increases when species are aggregated together into assemblages with more species, compared with the predictability of individual taxa. However, predictability within dinoflagellates and larger phytoplankton (>12 μm cell radius) is low overall and does not increase by aggregating similar species together. High variability in the data, due to observational error (noise) or stochasticity in population growth rates, reduces the predictability of individual species more than the predictability of assemblages. These findings show that there is greater potential for univariate prediction of species assemblages or whole‐community metrics, such as total chlorophyll or biomass, than for the individual dynamics of phytoplankton species.     

Mammalian adipogenesis regulator (Areg) cells use retinoic acid signalling to be non‐ and anti‐adipogenic in age‐dependent manner

Adipose stem and precursor cells (ASPCs) give rise to adipocytes and determine the composition and plasticity of adipose tissue. Recently, several studies have demonstrated that ASPCs partition into at least three distinct cell subpopulations, including the enigmatic CD142⁺ cells. An outstanding challenge is to functionally characterise this population, as discrepant properties, from adipogenic to non‐ and anti‐adipogenic, have been reported for these cells. To resolve these phenotypic ambiguities, we characterised mammalian subcutaneous CD142⁺ ASPCs across various experimental conditions, demonstrating that CD142⁺ ASPCs exhibit high molecular and phenotypic robustness. Specifically, we find these cells to be firmly non‐ and anti‐adipogenic both in vitro and in vivo, with their inhibitory signals also impacting adipogenic human cells. However, these CD142⁺ ASPC‐specific properties exhibit surprising temporal phenotypic alterations, and emerge only in an age‐dependent manner. Finally, using multi‐omic and functional assays, we show that the inhibitory nature of these adipogenesis‐regulatory CD142⁺ ASPCs (Aregs) is driven by specifically expressed secretory factors that cooperate with the retinoic acid signalling pathway to transform the adipogenic state of CD142⁻ ASPCs into a non‐adipogenic, Areg‐like state.     

First record of non‐flying mammalian contributors to pollination in a tropical montane forest in Asia

This study aims to identify the flower visitors of Mucuna thailandica (Fabaceae), endemic plant species in montane forests in Thailand, to determine their potential pollinators. The genus Mucuna produces papilionaceous flowers and has an explosive flower‐opening step. Explosive opening rapidly exposes stamens and pistil from keel petals and releases pollen. The flower of this species depends completely on animals to perform this step, essential for pollination success. Using a camera trap survey, we revealed that non‐flying mammals, such as squirrels (Callosciurus sp.) and masked palm civets (Paguma larvata), opened flowers explosively. Thus, these mammals contribute to the pollination of M. thailandica. This is the first report of non‐flying mammals contributing to pollination in montane forests in tropical Asia.     

A phase I clinical trial of human embryonic stem cell‐derived retinal pigment epithelial cells for early‐stage Stargardt macular degeneration: 5‐years' follow‐up

ObjectivesTo evaluate the long‐term biosafety and efficacy of transplantation of human embryonic stem cells‐derived retinal pigment epithelial (hESC‐RPE) cells in early‐stage of Stargardt macular degeneration (STGD1).Materials and methodsSeven patients participated in this prospective clinical study, where they underwent a single subretinal transplantation of 1 × 10⁵ hESC‐RPE cells in one eye, whereas the fellow eye served as control. These patients were reassessed for a 60‐month follow‐up through systemic and ophthalmic examinations.ResultsNone of the patients experienced adverse reactions systemically or locally, except for two who had transiently high intraocular pressure post‐operation. Functional assessments demonstrated that all of the seven operated eyes had transiently increased or stable visual function 1‐4 months after transplantation. At the last follow‐up visit, two of the seven eyes showed visual function loss than the baseline; however, one of them showed a stable visual acuity when compared with the change of fellow eye. Obvious small high reflective foci in the RPE layer were displayed after the transplantation, and maintained until the last visit. Interestingly, three categories of patients who were classified based on autofluorescence, exhibited distinctive patterns of morphological and functional change.ConclusionsSubretinal transplantation of hESC‐RPE in early‐stage STGD1 is safe and tolerated in the long term. Further investigation is needed for choosing proper subjects according to the multi‐model image and function assessments.     

Climate and habitat configuration limit range expansion and patterns of dispersal in a non‐native lizard

Invasive species are one of the main causes of biodiversity loss worldwide. As introduced, populations increase in abundance and geographical range, so does the potential for negative impacts on native communities. As such, there is a need to better understand the processes driving range expansion as species become established in recipient landscapes. Through an investigation into capacity for population growth and range expansion of introduced populations of a non‐native lizard (Podarcis muralis), we aimed to demonstrate how multi‐scale factors influence spatial spread, population growth, and invasion potential in introduced species. We collated location records of P. muralis presence in England, UK through data collected from field surveys and a citizen science campaign. We used these data as input for presence‐background models to predict areas of climate suitability at a national‐scale (5 km resolution), and fine‐scale habitat suitability at the local scale (2 m resolution). We then integrated local models into an individual‐based modeling platform to simulate population dynamics and forecast range expansion for 10 populations in heterogeneous landscapes. National‐scale models indicated climate suitability has restricted the species to the southern parts of the UK, primarily by a latitudinal cline in overwintering conditions. Patterns of population growth and range expansion were related to differences in local landscape configuration and heterogeneity. Growth curves suggest populations could be in the early stages of exponential growth. However, annual rates of range expansion are predicted to be low (5–16 m). We conclude that extensive nationwide range expansion through secondary introduction is likely to be restricted by currently unsuitable climate beyond southern regions of the UK. However, exponential growth of local populations in habitats providing transport pathways is likely to increase opportunities for regional expansion. The broad habitat niche of P. muralis, coupled with configuration of habitat patches in the landscape, allows populations to increase locally with minimal dispersal.     

Disturbance and the (surprising?) role of ecosystem engineering in explaining spatial patterns of non‐native plant establishment

Different conceptions of disturbance differ in the degree to which they appeal to mechanisms that are general and equivalent, or species‐, functional group‐, or interaction‐specific. Some concepts of disturbance, for example, predict that soil disturbances and herbivory have identical impacts on species richness via identical mechanisms (reduction in biomass and in competition). An alternative hypothesis is that the specific traits of disturbance agents (small mammals) and plants differentially affect the richness or abundance of different plant groups. We tested these hypotheses on a degu (Octodon degus) colony in central Chile. We ask whether native and non‐native forbs respond differently to degu bioturbation on runways versus herbivory on grazing lawns. We ask whether this can explain the increase in non‐native plants on degu colonies. We found that biopedturbation did not explain the locations of non‐native plants. We did not find direct evidence of grazing increasing non‐native herbs either, but a grazing effect appears to be mediated by grass, which is the dominant cover. Further, we provide supplementary evidence to support our interpretation that a key mechanism of non‐native spread is the formation of dry soil conditions on grazing lawns. Thus, ecosystem engineering (alteration of soil qualities) may be an outcome of disturbances, in which each interacts with specific plant traits, to create the observed pattern of non‐native spread in the colony. Based on these results, we propose to extend Jentsch and White (Ecology, 100, 2019, e02734) concept of combined pulse/ disturbance events to the long‐term process duality of ecosystem engineering/ disturbance.     

“Cancer Reviews 2021” series: Dynamic EMT: a multi‐tool for tumor progression

The process of epithelial–mesenchymal transition (EMT) is fundamental for embryonic morphogenesis. Cells undergoing it lose epithelial characteristics and integrity, acquire mesenchymal features, and become motile. In cancer, this program is hijacked to confer essential changes in morphology and motility that fuel invasion. In addition, EMT is increasingly understood to orchestrate a large variety of complementary cancer features, such as tumor cell stemness, tumorigenicity, resistance to therapy and adaptation to changes in the microenvironment. In this review, we summarize recent findings related to these various classical and non‐classical functions, and introduce EMT as a true tumorigenic multi‐tool, involved in many aspects of cancer. We suggest that therapeutic targeting of the EMT process will—if acknowledging these complexities—be a possibility to concurrently interfere with tumor progression on many levels.     

Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery

The Gram‐negative bacterium Legionella pneumophila is the causative agent of Legionnaires'' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella‐containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non‐virulent and a non‐replicating, virulent/transmissive phase. Here, we show on a single‐cell level that at late stages of infection, individual motile (P_flaA‐GFP‐positive) and virulent (P_ralF‐ and P_sidC‐GFP‐positive) L. pneumophila emerge in the cluster of non‐growing bacteria within an LCV. Comparative proteomics of P_flaA‐GFP‐positive and P_flaA‐GFP‐negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the P_flaA‐GFP‐positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi‐phasic life cycle of L. pneumophila.     

Structure‐based analysis and evolution of a monomerized red‐colored chromoprotein from the Olindias formosa jellyfish

GFP‐like chromoproteins (CPs) with non‐fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non‐fluorescent monomeric red CP, R‐Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R‐Velour at a 2.1 Å resolution. R‐Velour has a trans‐chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R‐Velour has a large torsion inducing non‐fluorescence property. Through site‐directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four‐color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus–Hush model, supports the spectrum shift of these mutants. These findings may support further development of R‐Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging.     

Co‐production of biofuel,bioplastics and biochemicals during extended fermentation of Halomonas bluephagenesis

Halomonas bluephagenesis TD1.0 was engineered to produce the biofuel propane, bioplastic poly‐3‐hydroxybutyrate (PHB), and biochemicals mandelate and hydroxymandelate in a single, semi‐continuous batch fermentation under non‐sterile conditions. Multi‐product separation was achieved by segregation of the headspace gas (propane), fermentation broth ([hydroxy]mandelate) and cellular biomass (PHB). Engineering was performed by incorporating the genes encoding fatty acid photodecarboxylase (CvFAP) and hydroxymandelic acid synthase (SyHMAS) into a H. bluephagenesis hmgCAB cassette knockout to channel flux towards (hydroxy)mandelate. Design of Experiment strategies were coupled with fermentation trials to simultaneously optimize each product. Propane and mandelate titres were the highest reported for H. bluephagenesis (62 g/gDCW and 71 ± 10 mg/L respectively) with PHB titres (69% g/gDCW) comparable to other published studies. This proof‐of‐concept achievement of four easily separated products within one fermentation is a novel achievement probing the versatility of biotechnology, further elevating H. bluephagenesis as a Next Generation Industrial Biotechnology (NGIB) chassis by producing highly valued products at a reduced cost.

Halomonas bluephagenesis TD1.0 was engineered to generate multiple products propane, poly‐3‐hydroxybutyrate and (hydroxy)mandate. These compounds are easily purified due to their location in the gas, phase, cell pellet and culture supernatant, respectively. This proof of principle study shows the potential application of multi‐product biosynthesis within an industrially relevant host as a route to renewable and sustainable bio manufacturing.     

The human GID complex engages two independent modules for substrate recruitment

The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi‐subunit complex remain poorly understood. Using biochemical assays, cross‐linking mass spectrometry, and cryo‐electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N‐end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8β isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo‐EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring‐like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non‐overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity.     

Two‐step nuclear centring by competing microtubule‐ and actin‐based mechanisms in 2‐cell mouse embryos

Microtubules typically promote nuclear centring during early embryonic divisions in centrosome‐containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time‐lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule‐dependent manner. High cytoplasmic viscosity in 2‐cell embryos is associated with non‐diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro‐centring actin‐dependent mechanisms work against microtubule‐dependent de‐centring forces. Disrupting the equilibrium of this tug‐of‐war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule‐dependent de‐centring forces.