In vitro action of aureomycin and terramycin on Balantidium coli

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1. Aureomycin produced inhibition of B. coli in vitro beginning at 0.4 mg/ml concentration.     

Aspects of the carbohydrate metabolism of a mutant of Neurospora crassa requiring acetate for growth

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1. Two mutant genes controlling the activities of different enzyme systems in Neurospora are described. One controls the activity of the enzyme pyruvic carboxylase, the other an enzyme system involved in the oxidation of pyruvic acid.     

Studies on cestode metabolism. II. The utilization of glycogen by Schistocephalus solidus in vitro

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1. The amount of endogenous glycogen used by Schistocephalus plerocercoids, when cultured to maturity, has been measured in a range of media and physical conditions.     

Comparison of Effects of Trichuris muris and Spontaneous Colitis on the Proximal Colon Microbiota in C3H/HeJ and C3Bir IL10–/– Mice

The nematode Trichuris muris has been shown to interact with specific enteric bacteria, but its effects on the composition of its host''s microbial community are not fully understood. We hypothesized that Trichuris muris-infected mice would have altered colon microbiota as compared with uninfected mice. Colon histopathology and microbial community structure and composition were examined in mouse models of colitis (C3BirTLR4^−/− IL10^−/− and C3H/HeJ TLR4^−/− IL10^+/+ mice) with and without T. muris infection, in uninfected C3BirIL10^−/− mice with and without spontaneous colitis, and in normal C3H/HeJ mice. T. muris-infected mice developed colon lesions that were more severe than those seen in IL10-deficient mice. Approximately 80% of infected IL10^−/− mice had colon neutrophilic exudates, and some had extraintestinal worms and bacteria. The composition and structure of proximal colon microbiota were assessed by using terminal restriction fragment length polymorphism analysis targeting the bacterial 16S rRNA gene. Colon microbiota in C3BirIL10^−/− and C3H/HeJ mice differed both qualitatively and quantitatively. Trichuris infection significantly altered the relative abundance of individual operational taxonomic units [OTU] but not the composition (presence or absence of OTU) of colon microbiota in the 2 mouse genotypes. When C3BirIL10^−/− and C3H/HeJ mouse OTU were considered separately, Trichuris was found to affect the microbiota of C3BirIL10^−/− mice but not of C3H/HeJ mice. Even though 34 of the 75 (45%) C3BirIL10^−/− mice had spontaneous colitis, neither qualitative nor quantitative differences were detected in microbiota between colitic or noncolitic C3BirIL10^−/− mice or noncolitic C3H/HeJ mice. Therefore, Trichuris-infected mice developed distinct microbial communities that were influenced by host background genes; these alterations cannot be attributed solely to colonic inflammation.roup method with arithmetic averaging; OTU, operational taxonomic unit; qPCR, quantitative real-time PCR; SIMPER, similarity percentage; T-RFLP, terminal restriction fragment length polymorphism

Trichuris spp. are gastrointestinal nematodes that dwell in close association with a complex bacterial community in the host''s colon. After ingestion, embryonated eggs hatch in the cecum or colon releasing first-stage larvae that penetrate the epithelium and undergo 4 molts before becoming sexually mature. Both larval and adult Trichuris form syncytial tunnels in the colonic epithelium^21,30 that anchor the organisms in the proximal colon, where females produce eggs that pass in feces and embryonate in the environment.T. suis excretory secretory products (ESP) condition the colonic environment for enhanced worm survival, including effects on intestinal bacteria. Previous work demonstrated that T. suis ESP had dose-dependent effects on the tight junctions of epithelial cells.¹ The ESP fraction below a molecular weight of 10,000 kDa was mainly composed of an antimicrobial moiety² with bactericidal activity against gram-negative (Campylobacter jejuni, C. coli, and Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria. In addition, due to several enzymatic activities, T. suis ESP have been demonstrated to aid the worms in burrowing into the host''s colonic epithelium and in feeding.^1,10,12 In addition to a 20-kDa diagnostic antigen,^10,11 higher molecular-weight fractions of ESP harbored a 42-kDa zinc metalloprotease that likely functions to provide nutrition for the worms through collagenase and elastase activities.¹⁰ Furthermore, a serine protease inhibitor (TsCEI) was purified from adult-stage T. suis by using acid precipitation, affinity chromatography, and reverse-phase HPLC.³³ This 6.43-kDa TsCEI inhibited chymotrypsin, pancreatic elastase, neutrophil elastase, and cathepsin G and was suggested to function as a parasite defense mechanism by modulating host immune responses. Indeed, exposure of cultured epithelial cells to T. suis ESP elicited IL6 and IL10 cytokine responses.³¹Trichuris has also been reported to interact with bacteria in vivo. Early studies demonstrated development of diarrhea in weaning age pigs concurrently harboring T. suis and various bacteria.³⁵ A mixed inoculum of T. suis and cecal scrapings containing Brachyspira, Campylobacter spp., or Salmonella spp. were implicated in this diarrhea by means of passive transfer to SPF pigs.³⁵ Interactions between this helminth and enteric bacteria were also explored by antibiotic treatment of T. suis-infected pigs.^20,27 Results of both passive transfer and antibiotic treatment experiments in pigs showed that Trichuris and various bacterial strains were necessary to produce the type of diarrhea and colonic lesions seen in weaning aged pigs in production, but did not implicate a single bacterial agent. In 2003, synergism between T. suis and C. jejuni was proven to cause mucohemorrhagic colitis in that germ-free piglets inoculated with both agents developed disease, whereas those infected with a single agent did not.²⁵ Recent studies in T. suis-infected pigs show changes in the microbial community of the colon with some accompanying metabolic changes.^22,45 Similar interactions have been found in extensive studies of captive rhesus monkeys with chronic enterocolitis. In these monkeys, severe disease was associated with presence of Trichuris trichiura and several enteric pathogens including C. coli, C. jejuni, Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni.³⁸ Therefore, Trichuris interacts with and may demonstrate synergy in disease production with the host''s colonic microflora.Interactions between Trichuris and bacteria have also been studied in mice.^9,20,36 One study found 100% morbidity in C57BL/6 IL10^−/− and congenic IL10^−/− IL4^−/− mice after challenge with T. muris.³⁶ The authors hypothesized that this high morbidity was due to an overgrowth of opportunistic invasive bacteria that use the mechanical damage caused by T. muris larvae to breach the intestinal tract. Adding the broad-spectrum antibiotic neomycin sulfate to the drinking water of IL10^−/− IL4^−/− mice and then infecting them with T. muris resulted in a statistically significant increase in the percentage of mice that survived infection.³⁶ The authors concluded that growth of opportunistic bacteria may have contributed to the previously observed morbidity and mortality. Most recently, another group⁹ found that increased levels of colonic microflora favor increased numbers of T. muris and chronic infections. The group also demonstrated that T. muris eggs hatched more efficiently in vitro when incubated with explants of mouse cecum containing 5 isolates of bacteria (E. coli, Staphylococcus aureus, Salmonella typhimurium, or Pseudomonas aeruginosa) and the yeast Saccharomyces cerevisiae, with the greatest effects seen at 37 °C. Similarly, work from our laboratory²⁰ demonstrated that treatment of T. muris-infected C57BL/6 IL10^−/− mice with metronidazole but not prednisolone increased survival.²⁰ Most recently, chronic infections with T. muris in C57BL/6 mice have been shown to decrease the diversity of intestinal microbiota,¹³ increase the abundance of Lactobacillus spp., and alter the metabolome.¹⁴Taken together, these data suggest an important microbial component to the pathogenesis of Trichuris infections in a variety of species. Given that Trichuris suis has been administered to patients with inflammatory bowel disease (IBD), and in some studies appeared to diminish IBD symptoms^42,43 we sought to understand the community-wide interactions of this worm with enteric bacteria in a mouse model of colitis. We hypothesized that the microbiota of the proximal colon would differ significantly in mice infected with T. muris as compared with uninfected mice. We theorized that these effects would occur due to the worm''s immunomodulatory properties in the host and may contribute to the successful outcomes of Trichuris treatment in patients with IBD.     

Healthy herds in the phytoplankton: the benefit of selective parasitism

The impact of selective predation of weaker individuals on the general health of prey populations is well-established in animal ecology. Analogous processes have not been considered at microbial scales despite the ubiquity of microbe-microbe interactions, such as parasitism. Here we present insights into the biotic interactions between a widespread marine thraustochytrid and a diatom from the ecologically important genus Chaetoceros. Physiological experiments show the thraustochytrid targets senescent diatom cells in a similar way to selective animal predation on weaker prey individuals. This physiology-selective targeting of ‘unhealthy’ cells appears to improve the overall health (i.e., increased photosynthetic quantum yield) of the diatom population without impacting density, providing support for ‘healthy herd’ dynamics in a protist–protist interaction, a phenomenon typically associated with animal predators and their prey. Thus, our study suggests caution against the assumption that protist–protist parasitism is always detrimental to the host population and highlights the complexity of microbial interactions.Subject terms: Microbial ecology, Water microbiology

Animal predators can exert overall positive effects on the health of prey populations by removing individuals with suboptimal health [1, 2] in a manner that has been termed ‘healthy herd’ dynamics [3]. While such top-down processes are well-established in animal ecology [1–3], they have largely been unconsidered in microbe-microbe interactions.Protist–protist parasitism is widespread in the marine environment [4] and is generally considered to be detrimental to host populations [5, 6]. However, despite their ubiquity, the ecophysiological impact of protist–protist parasitism remains poorly understood. An important case that necessitates investigation is protist parasitism of diatoms, which have limited representation with culture-dependent model systems despite the significance of diatoms in marine ecosystem functioning and global primary production [7].We observed and isolated a heterotrophic protist growing epibiotically on moribund and dead Chaetoceros sp. diatoms from a summer bloom at Station L4 in the Western English Channel off Plymouth (UK) (Fig. 1A, B; Supplementary Figs. 1 and 2; Supplementary Methods). Single-cell picking achieved diatom and parasite co-cultures and uninfected host diatoms. The 18 S rRNA gene V4 region of the protist (termed ‘ThrauL4’) identified the epibiont as a novel thraustochytrid (Stramenopila; Labyrinthulomycota; Thraustochytrida) (Supplementary Fig. 3). Searching for ThrauL4 18 S rRNA gene homologues in the Ocean Sampling Day dataset revealed that the parasite has a wide distribution in coastal temperate regions (Supplementary Fig. 4).Open in a separate windowFig. 1Growth experiments demonstrate that thraustochytrids preferentially target and grow on unhealthy diatom cells.A Differential interference contrast (DIC) image of Chaetoceros chain exhibiting different degrees of infection by ThrauL4. Uninfected cell (un), a lightly infected cell (li), heavily infected cells (hi) and a dead, empty frustule (d). Scale bar = 20 µm. B Scanning Electron Micrograph (SEM) of a Chaetoceros diatom swarmed by ThrauL4. Scale bar = 5 µm. C ThrauL4 growth dynamics on a selected range of diatoms and dinoflagellates (Alexandrium minutum and Prorocentrum minimum) (±SEM, n = 3). D Chaetoceros growth with ThrauL4 (±SEM, n = 5). Dashed lines demarcate the lag (1), exponential (2) and stationary (3) phases of Chaetoceros growth. E Time-lapse of Chaetoceros-ThrauL4 showing ThrauL4 colonising unhealthy cells. Asterisk = cytoplasmic bleb from unhealthy diatom. Arrowhead = initial thraustochytrid colonisation. Timestamp = HH:MM:SS. Difference in the abundance (F) and prevalence (G) of parasites in healthy (control), stressed and dead Chaetoceros populations (n = 5) inoculated with ThrauL4 following heat stress exposure. ANOVA Tukey’s HSD n.s p > 0.05 (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. H Example diatom exposed to different laser powers used to generate individual Chaetoceros cells of varying health. Red channel overlay demarks chlorophyll autofluorescence. Scale bar = 5 µm. I Time taken for individual diatom cells (n = 15) exposed to varying laser treatments to be colonised by ThrauL4. J Diagrammatic representation of the proposed diatom-thraustochytrid interaction cycle based on time-lapse microscopy observations (see Supplementary Videos).Stable Chaetoceros-ThrauL4 co-cultures permitted the characterisation of ThrauL4 internal structures (Supplementary Figs. 5 and 6), epibiotic growth (Fig. 1A, B; Supplementary Figs. 7 and 8) and infection dynamics (Fig. 1C, D). ThrauL4 also attached to other diatoms (Odontella sinensis, Ditylum brightwellii and Coscindodiscus sp.) in a similar manner to Chaetoceros sp. but not dinoflagellates (Fig. 1C; Supplementary Fig. 9).The proportion of diatom cells with ThrauL4 attached increased when Chaetoceros sp. cells entered the stationary growth phase (Fig. 1D). Time-lapse microscopy revealed the dynamic nature of the ThrauL4-diatom interaction (Fig. 1E, Supplementary Movies 1–6), with the motile ThrauL4 apparently targeting physiologically ‘unhealthy’ cells identified by cytoplasmic blebbing prior to colonisation (Fig. 1E).We set out to test the hypothesis that ThrauL4 targeted unhealthy diatoms using population-level ecophysiology experiments. When introduced to heat-stressed diatom populations, ThrauL4 had a higher fitness (i.e. became more abundant) and infected more Chaetoceros sp. cells than when exposed to healthy un-stressed diatoms (Fig. 1F, G), confirming more optimal growth of the parasite amongst unhealthy diatom populations. Furthermore, selective targeting was also demonstrated at the single-cell level using laser-damaged individual cells and time-lapse microscopy (Fig. 1H, I). 80% of stressed cells and 60% of dead cells were colonised by ThrauL4 during the 30 min experimental period, whereas diatoms in healthy control populations were un-colonised.These results led us to investigate the physiological impact of thraustochytrid parasitism on host diatom populations by comparing the dynamics and health of parasite exposed and non-exposed Chaetoceros sp. populations (Fig. 2A–C). Based on the previous growth experiments showing ThrauL4 proliferation during the diatom stationary phase (Fig. 1D), Chaetoceros sp. cultures grown to their stationary phase after 7 d were chosen to mimic environmental bloom decline. Using the photosynthetic quantum yield (Fv/Fm) as a proxy for overall diatom health [8], after 8 d, the parasitized Chaetoceros sp. populations were consistently healthier than those in the control non-exposed populations (Fig. 2A). Diatom population density was similar in both treatments (Fig. 2B) and parasite prevalence peaked after 8 days (Fig. 2C). In a separate experiment to investigate the role of genotype specificity in ThrauL4 parasitism, we generated a clonal Chaetoceros sp. population by single-cell picking and exposed the population to ThrauL4 cultures growing independently from diatoms. Although the clonal population declined in health more rapidly overall, ThrauL4 parasitism also resulted in healthier populations (Fig. 2D–F) suggesting that these results are a not an artefact of genotype specificity and succession.Open in a separate windowFig. 2Selective targeting of unhealthy diatom cells by thraustochytrids improves the overall health of the diatom population.A–C Population dynamics of the Fv/Fm (A) and total number (B) of stationary Chaetoceros diatoms for control and parasitized diatom populations over the experimental period (±SEM, n = 5). Welch’s t-test *p < 0.05, **p < 0.01, ***p < 0.001. The parasite prevalence did not exceed about a third of the total population (C) (±SEM, n = 5). Parasites added at 0 d. In a separate experiment (D–F), a clonal Chaetoceros population was generated. Population dynamics of the Fv/Fm (D), total number (E) and infection prevalence (F) of stationary Chaetoceros diatoms for control and parasitized populations made clonal by single-cell picking (±SEM, n = 5). Significance values as above. Parasites added at 0 day. Taken together these results indicate that preferential thraustochytrid parasitism of unhealthy diatoms strengthens the overall health of the population therefore providing evidence for the ‘healthy herd’ hypothesis in a phytoplankton population, which is summarised diagrammatically in (G).By removing physiologically weaker individuals from the population, the remaining cells will constitute an overall healthier population. However, other mechanisms may also promote an overall healthier diatom population. It may be that selective parasitism relieves nutrient competition between unhealthy and healthy individuals. In the natural environment, diatom-diatom competition is a major growth limiting factor [9, 10] and removing the pressure exerted by weaker cells may allow the population to be more robust. It is also possible that the thraustochytrid could be ‘cleaning’ the population by preventing the build-up of toxic waste products or the proliferation of detrimental co-culture bacteria in an analogous way to how carrion removal by vultures prevents the spread of diseases to mammals [11]. In addition, thraustochytid parasitism could accelerate nutrient recycling by releasing nutrients from dying cells. The consequences of physiology-selective diatom parasitism should be assessed in the marine environment, including impacts at the community scale and in the context of ecosystem functioning.The proposed influence of thraustochytrid parasitism on diatom population health is summarised in Fig. 2G. We suggest that this thraustochytrid-diatom interaction provides evidence of ‘healthy herd’ dynamics in a protist–protist interaction, an ecological phenomenon typically associated with animal predator-prey interactions [3]. As we show here with ThrauL4, animal predators such as lions [12], cougars [13], African wild dogs [14], and wolves [15] have been shown to target prey with suboptimal health. The ‘healthy herd’ hypothesis states that by selective predation on unhealthy prey, predators increase the overall health of the prey population by increasing resource availability or by removing potential carriers of disease [3]. Evidence for ‘healthy herd’ dynamics where predation generates healthier prey populations has also been demonstrated in lobster-sea urchin [16], fish-Daphnia [17], and fox-grouse [18] predator–prey systems. Here, we provide analogous supportive evidence from a marine protist–protist system.‘Heathy herd’ dynamics between protists challenges the assumption that protist–protist parasitism is always detrimental to the host population and raises caution in this assumption in ecosystem modelling or inference from molecular ecology surveys (e.g., metabarcoding). Our results have demonstrated the potential complexity of protist–protist symbioses, highlighting the value of culture-based experimentation and the importance of developing model co-culture systems in resolving complex ecological interactions. The underpinning biology and ecological importance in natura of such interactions now require further investigation.     

The Aspergillus fumigatus Secretome Alters the Proteome of Pseudomonas aeruginosa to Stimulate Bacterial Growth: Implications for Co-infection

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Highlights

• •Pseudomonas aeruginosa growth increases in Aspergillus fumigatus culture filtrates.
• •A. fumigatus culture filtrates are characterized by a range of peptidases and proteases.
• •LFQ proteomics characterizes the response of P. aeruginosa to A. fumigatus culture filtrates.
• •A. fumigatus creates an environment for P. aeruginosa to proliferate.

     

Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice

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Highlights

• •db/db β-cells restores appropriate insulin stores and normalize secretory function.
• •Numerous changes in the phosphorylation and sialylation states by euglycemic rest.
• •Restoration of numerous dysfunctional biological processes following euglycemic rest.
• •β-cell adaptive flexibility may lead to improvement in endogenous β-cell function.

     

Membrane protein biogenesis by the EMC

Recent cryo‐EM‐based models reveal how the ER membrane protein complex may accomplish insertion of protein transmembrane domains with limited hydrophobicity.

Insertion of strongly hydrophobic TMDs into the ER membrane is mediated by the Sec61 complex for co‐translational insertion and the GET complex for post‐translational insertion of tail‐anchors (Volkmar & Christianson, 2020). By contrast, the EMC inserts TMDs of limited hydrophobicity, frequently located at the N‐ or C‐termini of proteins, and is involved in biogenesis of multi‐spanning membrane proteins (Volkmar & Christianson, 2020).The EMC is highly conserved (Wideman, 2015). In vertebrates, ten subunits have been identified (EMC1‐10), two of which, EMC8 and EMC9, are homologous and the result of a vertebrate‐specific gene duplication (Wideman, 2015). In Saccharomyces cerevisiae, EMC8 has been lost (Wideman, 2015). Only EMC3 displays clear homology to other membrane protein insertases, the Oxa1 family (Wideman, 2015; Volkmar & Christianson, 2020). This family includes YidC, which inserts TMDs into the bacterial cytoplasmic membrane, usually in cooperation with the Sec61‐homologous SecYEG channel (Volkmar & Christianson, 2020). Their association, along with the SecDF ancillary complex, forms a holo‐translocon capable of protein secretion and TMD insertion, with striking similarities to the EMC complex (Martin et al, 2019).Recent work by Pleiner et al (2020) presented a 3.4 Å cryo‐EM structure of the human EMC purified via a GFP‐tag on EMC2 and incorporated into a phospholipid nanodisc. The complex is formed by nine proteins (EMC1‐8, EMC10) (Pleiner et al, 2020). EMC8 and EMC9 are structurally similar, and their association with EMC2 is mutually exclusive (O''Donnell et al, 2020). Of the 12 TMDs, nine constitute the pseudosymmetric central ordered core, with a basket‐shaped cytosolic vestibule formed primarily by alpha‐helices of the EMC3 and EMC6 TMDs and cytosolic EMC2 (Fig 1A; Pleiner et al, 2020). The L‐shaped lumenal domain of the EMC consists mostly of beta‐sheets (Fig 1A; Pleiner et al, 2020), flanked by a conspicuous and conserved amphipathic alpha‐helix of EMC1 sealing the vestibule at the interface between the membrane and the ER lumen, together with another smaller amphipathic helix contributed by EMC3 (Fig 1A; Pleiner et al, 2020). In the ER lumen, the two 8‐bladed propellers of EMC1 contact six of the eight other subunits and stabilize the entire complex (Fig 1A; Pleiner et al, 2020). Beta‐sandwiches of EMC7 and EMC10 are anchored to the EMC1 lumenal domain (Fig 1A; Pleiner et al, 2020). In the cytosol, the tetratricopeptide repeat (TPR) spiral of EMC2 forms a cup underneath the partially hydrophilic vestibule in the membrane between the TMDs of EMC3 and EMC6, bridging the cytosolic ends of TMDs of EMC1, 3 and 5 (Fig 1A; Pleiner et al, 2020). Cytosolic EMC8 is bound to the opposite face of EMC2 (Fig 1A).Open in a separate windowFigure 1Comparison of the structures of human and yeast EMC(A) Cryo‐EM 3D map of the human (emdb‐21929) and yeast (emdb‐21587) EMC, showing front and back views with individual subunits coloured. Membrane position, obtained from the OPM database, is shown by grey discs. (B) Close‐up view of the EMC cavity formed by EMC3 and EMC6. Left, shown in a hydrophobicity surface pattern. Right, surface representation overlapped with the TMDs of EMC3 and EMC6. EMC4, flexible and with a gate function at the substrate‐binding place, is shown in pink in the yeast representation. EMC4 is not visible at the atomic EMC human structure, although is observed as a weak density at the human model, accompanied by TMs of EMC7 and EMC10 (Pleiner et al, 2020). (C) The yeast EMC following > 5 µs of CG‐MD simulation. The protein is shown as surface and coloured as per Pleiner et al (2020). The computed densities of waters and phospholipid tails and phosphates are shown as blue, yellow and lime green densities, sliced to bisect the cavity for clarity. Right, inset of the EMC cavity. Methods: CG‐MD simulations were built using PDB 6WB9 in a solvated symmetric POPC/POPE/cholesterol membrane and run in the Martini forcefield as described in Martin et al (2019). 3 µs unrestrained simulations were run, followed by 2.5 µs backbone restrained simulation for density calculation, done using VolMap in VMD (Humphrey et al, 1996).The 3.0 Å cryo‐EM structure of the yeast EMC presented by Bai and colleagues shows a very similar overall organization (Bai et al, 2020). Here, purification was via a 3xFLAG‐tag on EMC5, and the structure of the 8‐subunit complex (without EMC8/9) was visualized in detergent solution (Bai et al, 2020). The yeast complex has twelve TMDs like the human EMC, but unlike the human structure, EMC4 in yeast has three TMDs that are clearly visible (Bai et al, 2020). They are angled in the membrane pointing away from the complex at the cytosolic end (Fig 1A), and Bai et al (2020) propose that TMDs of EMC4, EMC3 and EMC6 form a substrate‐binding pocket similar to that of YidC. As in the human EMC, there are two amphipathic helices (EMC1 and EMC3) at the membrane/lumen interface (Fig 1A; Bai et al, 2020). In the ER lumen, yeast EMC1 only has one 8‐bladed beta‐propeller, to which the beta‐sandwiches of EMC7 and EMC10 are anchored (Fig 1A; Bai et al, 2020). In the cytosol, EMC2 bridges EMC3, 4 and 5, and its TPR repeats form a cup underneath the vestibule similar to human EMC2 (Fig 1A; Bai et al, 2020).The authors propose that insertion of a partially hydrophilic TMD by the yeast EMC is mechanistically similar to insertion by bacterial YidC (Bai et al, 2020). Yeast EMC is proposed to bind substrate between TMD2 of EMC3 and TMD2 of EMC4 in a pocket with polar and positively charged amino acids at either end and hydrophobic amino acids in the centre (Fig 1B; Bai et al, 2020). Much has been made of a conserved positive region within the EMC complex here, present in an equivalent position also in YidC (Kumazaki et al, 2014): It is claimed to be important for the incorporation of more‐hydrophilic TMDs and perhaps responsible for the “positive‐inside” orientation rule (von Heijne, 1992). Yeast and human EMC3 contain a specific R31 and R26 residue, respectively, conserved also in YidC and important for function of the EMC, as well as for YidC in Gram‐positive, but interestingly not Gram‐negative, bacteria (Chen et al, 2014; Pleiner et al, 2020; Bai et al, 2020). Another interesting feature, also conserved with YidC, is the flexibility of the TMDs flanking the substrate‐binding pocket, critical for EMC entry of substrates (Bai et al, 2020).In the human EMC, methionine residues in a cytosolic loop of EMC3 act as a substrate bait (Pleiner et al, 2020). Polar and charged residues within the substrate‐binding groove guide the lumenal domain across the membrane, facilitated by local membrane thinning (Pleiner et al, 2020; Fig 1B). The positive charges within the substrate‐binding site exclude signal peptides and enforce the “positive‐inside rule” (von Heijne, 1992; Pleiner et al, 2020). Flexible TMDs of EMC4, EMC7 and EMC10 forming a “lateral gate” of the substrate‐binding groove allow sampling of the bilayer by the substrate TMD (Pleiner et al, 2020). As the shortened TMDs of EMC3 and EMC6 cannot stably bind the substrate TMD, they favour its release into the bilayer (Pleiner et al, 2020). The EMC1 beta‐propeller(s) may recruit additional protein maturation factors in the ER lumen (Pleiner et al, 2020; Bai et al, 2020) or bind the Sec61 channel to allow cooperation between the two insertases (Bai et al, 2020).Arguably, the most interesting feature of the EMC complex is the location of a large interior cavity with distinctive hydrophilic character, which likely aids TMD insertion (Fig 1B). We ran a coarse‐grained molecular dynamics (CG‐MD) simulation of the yeast EMC structure, which highlights a profound perturbation of the phospholipid bilayer in the EMC interior cavity (Fig 1C). Here, a deep gorge forms in the cytoplasmic leaflet of the bilayer, allowing the cavity to become flooded with water (Fig 1C). Note the location of the lipid head groups here (lime green), which presumably define the site of amphipathic TMD insertion. The incursion of phospholipids into the centre of the EMC complex is a feature shared by the bacterial holo‐translocon (Martin et al, 2019) and perhaps by all membrane protein insertases. The shape and character of the EMC cavity presumably dictate its predisposition for less hydrophobic TMDs; it would be interesting to see whether the cavities of different insertases are similarly tailored to suit their substrates.