Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Cryo-EM structure of the RADAR supramolecular anti-phage defense complex.

RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.

Bacterial gasdermins reveal an ancient mechanism of cell death.

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.

Visualization of Fluoroscopic Imaging in Orthopedic Surgery: Head-Mounted Display vs Conventional Monitor.

Purpose. See-through head-mounted displays (HMDs) can be used to view fluoroscopic imaging during orthopedic surgical procedures. The goals of this study were to determine whether HMDs reduce procedure time, number of fluoroscopic images required, or number of head turns by the surgeon compared with standard monitors. Methods. Sixteen orthopedic surgery residents each performed fluoroscopy-guided drilling of 8 holes for placement of tibial nail distal interlocking screws in an anatomical model, with 4 holes drilled while using HMD and 4 holes drilled while using a standard monitor. Procedure time, number of fluoroscopic images needed, and number of head turns by the resident during the procedure were compared between the 2 modalities. Statistical significance was set at P < .05. Results. Mean (SD) procedure time did not differ significantly between attempts using the standard monitor (55 [37] seconds) vs the HMD (56 [31] seconds) (P = .73). Neither did mean number of fluoroscopic images differ significantly between attempts using the standard monitor vs the HMD (9 [5] images for each) (P = .84). Residents turned their heads significantly more times when using the standard monitor (9 [5] times) vs the HMD (1 [2] times) (P < .001). Conclusions. Head-mounted displays lessened the need for residents to turn their heads away from the surgical field while drilling holes for tibial nail distal interlocking screws in an anatomical model; however, there was no difference in terms of procedure time or number of fluoroscopic images needed using the HMD compared with the standard monitor.

Implementation of point-of-care blood gas testing at a large community hospital: Cost analysis, sepsis bundle compliance, and employee engagement.

INTRODUCTION/BACKGROUND: Point-of-care testing (POCT) platforms support patient-centered approaches to health care delivery and may improve patient care. We evaluated implementation of a POCT platform at a large, acute care hospital in the Midwestern United States. METHODS: We used lactate testing as part of a sepsis bundle protocol to evaluate compliance and mortality outcomes. Respiratory team members were surveyed to assess perception of efficiency, ease of use, timely patient care, and overall engagement with the POCT system. Annualized cost per test of a benchtop analyzer and a POCT platform were compared across 3 years for each platform. RESULTS: Lactate testing volume increased from 61% to 91%, which was associated with improved sepsis bundle protocol compliance. Employees reported high levels of engagement, improvements in efficiency and time savings, and better patient care with POCT. Average cost per test was $10.02 for the benchtop system and $6.21 for the POCT platform. POCT saved our institution $88,476 annually in labor costs. DISCUSSION: Combined with a robust training program emphasizing the use of lactate testing in the context of the overall clinical picture, POCT enabled adherence to the sepsis bundle protocol and may have contributed to lower mortality. Additionally, the COVID-19 pandemic has provided us with unanticipated benefits of using POCT; it has enhanced our ability to deal with stringent infectious disease protocols, saving time and minimizing patient and staff exposure. CONCLUSIONS: Implementation of a POCT platform was associated with improved compliance to our sepsis protocol, reduced sepsis mortality, high employee engagement, and cost savings.

TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level.

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, the balancer group-peptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. However, complementary ions of the popular TMT-tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTpro-tag, which increases complementary ion plexing capacity to eight channels (TMTproC). Furthermore, the beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC, resulting in ∼65% more proteins quantified compared to TMTpro-MS3 and ∼18% more when compared to real-time-search TMTpro-MS3 (RTS-SPS-MS3). TMTproC quantification is more accurate than TMTpro-MS2 and even superior to RTS-SPS-MS3. We provide the software for quantifying TMTproC data as an executable that is compatible with the MaxQuant analysis pipeline. Thus, TMTproC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

4-Amino-1,8-naphthalimide-ferrocene conjugates as potential multi-targeted anticancer and fluorescent cellular imaging agents.

Herein we present eight ferrocenyl 4-amino-1,8-naphthalimides. Designed as fluorescent logic gates for acidity and oxidisability, the molecules have been repurposed as anti-proliferation and cellular imaging agents. The compounds were studied in vitro against MCF-7 and K562 cancer cell lines by the MTT method. Compounds with protonable secondary amines tended to exhibit greater cytotoxicity than those with tertiary amines. Compounds with no measurable GI50 values within a 24 hour time window, as well as at shorter exposure times, may be suitable as fluorescent cellular imaging probes.

Development and Pre-Clinical Analysis of Spatiotemporal-Aware Augmented Reality in Orthopedic Interventions.

Suboptimal interaction with patient data and challenges in mastering 3D anatomy based on ill-posed 2D interventional images are essential concerns in image-guided therapies. Augmented reality (AR) has been introduced in the operating rooms in the last decade; however, in image-guided interventions, it has often only been considered as a visualization device improving traditional workflows. As a consequence, the technology is gaining minimum maturity that it requires to redefine new procedures, user interfaces, and interactions. The main contribution of this paper is to reveal how exemplary workflows are redefined by taking full advantage of head-mounted displays when entirely co-registered with the imaging system at all times. The awareness of the system from the geometric and physical characteristics of X-ray imaging allows the exploration of different human-machine interfaces. Our system achieved an error of 4.76 ± 2.91mm for placing K-wire in a fracture management procedure, and yielded errors of 1.57 ± 1.16° and 1.46 ± 1.00° in the abduction and anteversion angles, respectively, for total hip arthroplasty (THA). We compared the results with the outcomes from baseline standard operative and non-immersive AR procedures, which had yielded errors of [4.61mm, 4.76°, 4.77°] and [5.13mm, 1.78°, 1.43°], respectively, for wire placement, and abduction and anteversion during THA. We hope that our holistic approach towards improving the interface of surgery not only augments the surgeon's capabilities but also augments the surgical team's experience in carrying out an effective intervention with reduced complications and provide novel approaches of documenting procedures for training purposes.

A memory of eS25 loss drives resistance phenotypes.

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.

Ovarian Hormones and Reward Processes in Palatable Food Intake and Binge Eating.

Ovarian hormones are associated with risk for binge eating in women. Recent animal and human studies suggest that food-related reward processing may be one set of neurobiological factors that contribute to these relationships, but additional studies are needed to confirm and extend findings.

Head-Mounted Display Use in Surgery: A Systematic Review.

Purpose. We analyzed the literature to determine (1) the surgically relevant applications for which head-mounted display (HMD) use is reported; (2) the types of HMD most commonly reported; and (3) the surgical specialties in which HMD use is reported. Methods. The PubMed, Embase, Cochrane Library, and Web of Science databases were searched through August 27, 2017, for publications describing HMD use during surgically relevant applications. We identified 120 relevant English-language, non-opinion publications for inclusion. HMD types were categorized as "heads-up" (nontransparent HMD display and direct visualization of the real environment), "see-through" (visualization of the HMD display overlaid on the real environment), or "non-see-through" (visualization of only the nontransparent HMD display). Results. HMDs were used for image guidance and augmented reality (70 publications), data display (63 publications), communication (34 publications), and education/training (18 publications). See-through HMDs were described in 55 publications, heads-up HMDs in 41 publications, and non-see-through HMDs in 27 publications. Google Glass, a see-through HMD, was the most frequently used model, reported in 32 publications. The specialties with the highest frequency of published HMD use were urology (20 publications), neurosurgery (17 publications), and unspecified surgical specialty (20 publications). Conclusion. Image guidance and augmented reality were the most commonly reported applications for which HMDs were used. See-through HMDs were the most commonly reported type used in surgically relevant applications. Urology and neurosurgery were the specialties with greatest published HMD use.

eIF5B gates the transition from translation initiation to elongation.

Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.

Aminonaphthalimide hybrids of mitoxantrone and amonafide as anticancer and fluorescent cellular imaging agents.

Novel water-soluble 4-aminonaphthalimides were synthesised and their cellular fluorescent imaging, cytotoxicity and ability to induced apoptosis evaluated. The lead compound 1 was designed from the cross-fertilisation of the basic hydrophilic amino pharmacophore of mitoxantrone, and an aminonaphthalimide scaffold of the drug candidate, amonafide. The compounds are also fluorescent pH probes based on photoinduced electron transfer (PET) and internal charge transfer (ICT). The compounds are sensitive to solvent polarity with large Stoke shifts (>90 nm) and provide emissive-coloured solutions (blue to yellow). Excited state pKas of 9.0-9.3 and fluorescence quantum yields of 0.47-0.58 were determined in water. The cytotoxicity and cellular fluorescent imaging properties of the compounds were tested on human cancer cell lines K562 and MCF-7 by the MTT assay, phase contrast and fluorescence microscopy. Compounds 1 and 3 with flexible aminoalkyl chains exhibited GI50 comparable to amonafide, while 2 and 4 with a rigid piperazine moiety and butyl chain are less cytotoxic. Fluorescence microscopy with 1 allowed for the visualization of the intracellular microenvironment exemplifying the potential utility of such hybrid molecules as anticancer and fluorescent cellular imaging agents.

Muscle biopsies in clinical trials for Duchenne muscular dystrophy - Patients' and caregivers' perspective.

The number of clinical trials for Duchenne muscular dystrophy is increasing. Many trials require muscle biopsies, which involve an invasive surgical procedure. Little is known about short- and long-term impacts of muscle biopsies as perceived by patients and caregivers. Therefore a survey was held among patients and their caregivers who participated in trials involving muscle biopsies, in seven countries. Seventy-eight responses were received. Analysis revealed that many patients and parents had significant anxiety before the biopsy. The main concern of caregivers was the required general anaesthesia. In most cases biopsies caused pain and temporarily hampered daily activities. The main long-term impact was scarring, although large variation in size was reported. Seventy-nine percent of caregivers were little bothered and 21% were moderately or severely bothered by the scar. Willingness to consider another biopsy in future protocols was higher for open-label studies than for placebo-controlled trials. Caregivers stressed the importance of knowing the results of biopsy analyses; only a minority actually received this information. Recommendations are made on the informed consent procedure regarding risks and consequences of muscle biopsies, and communication of results. Furthermore, efforts should be made to minimise the impact of biopsies through pain management and by considering plastic surgery.

Image-Guided Chondrocyte Harvesting for Autologous Chondrocyte Implantation: Initial Feasibility Study with Human Cadaver and Pilot Clinical Experience.

BACKGROUND: Autologous chondrocyte implantation (ACI), a promising modality for repairing full-thickness cartilage defects, requires 2 consecutive arthroscopic procedures for chondrocyte harvesting and implantation. In the present study, we assessed the feasibility and efficacy of image-guided chondrocyte harvesting as an alternative to arthroscopic biopsy. METHODS: We induced full-thickness cartilage defects in 10 human cadaveric knees. Computed tomographic arthrography (CTA) was performed following the intra-articular administration of Omnipaque 350 to measure the diameters of the induced cartilage defects. Subsequently, 2 independent operators conducted CTA-guided chondrocyte harvesting (from the medial and lateral trochlear ridges) in each knee. The time for chondrocyte harvesting, accuracy (distance between the predefined target on CTA and the final insertion site of the needle), and number of needle readjustments were recorded. In the institutional review board-approved clinical study, informed consent was obtained and chondrocyte harvesting was performed both with use of a conventional arthroscopic biopsy method and with use of a needle through an arthroscopy access site in 10 subjects for whom ACI was indicated. The samples were processed and cultured blindly, and the quantity and quality of the samples were determined. RESULTS: CTA measurements of full-thickness cartilage defects showed high to perfect absolute agreement and consistency when compared with direct measurements (overall interclass correlation coefficient, 0.933 to 0.983; p < 0.05). For both operators, image-guided chondrocyte harvesting from the lateral ridge was more accurate (p = 0.007 and 0.040) and faster (p = 0.056 and 0.014) in comparison with harvesting from the medial ridge. In the clinical study, no significant difference was observed for the growth index of samples between the needle-harvest and conventional methods (p = 0.897). CONCLUSIONS: CTA can be used for precise measurement of full-thickness cartilage defects. Image-guided chondrocyte harvesting is a viable alternative to traditional arthroscopic biopsy for ACI. CLINICAL RELEVANCE: We recognize the current pivotal role of arthroscopic biopsy, as a part of ACI, for chondrocyte harvesting as well as for delineating the nature of the lesion. However, on the basis of our results, image-guided chondrocyte retrieval may obviate the need for arthroscopic biopsy in some patients in the future.

RACK1 on and off the ribosome.

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein (RP) implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as a target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any RP from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular nonessentiality from yeast to humans, RACK1 readily reassociates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight into the biochemical basis of RPs exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

Interactive Flying Frustums (IFFs): spatially aware surgical data visualization.

PURPOSE: As the trend toward minimally invasive and percutaneous interventions continues, the importance of appropriate surgical data visualization becomes more evident. Ineffective interventional data display techniques that yield poor ergonomics that hinder hand-eye coordination, and therefore promote frustration which can compromise on-task performance up to adverse outcome. A very common example of ineffective visualization is monitors attached to the base of mobile C-arm X-ray systems. METHODS: We present a spatially and imaging geometry-aware paradigm for visualization of fluoroscopic images using Interactive Flying Frustums (IFFs) in a mixed reality environment. We exploit the fact that the C-arm imaging geometry can be modeled as a pinhole camera giving rise to an 11-degree-of-freedom view frustum on which the X-ray image can be translated while remaining valid. Visualizing IFFs to the surgeon in an augmented reality environment intuitively unites the virtual 2D X-ray image plane and the real 3D patient anatomy. To achieve this visualization, the surgeon and C-arm are tracked relative to the same coordinate frame using image-based localization and mapping, with the augmented reality environment being delivered to the surgeon via a state-of-the-art optical see-through head-mounted display. RESULTS: The root-mean-squared error of C-arm source tracking after hand-eye calibration was determined as [Formula: see text] and [Formula: see text] in rotation and translation, respectively. Finally, we demonstrated the application of spatially aware data visualization for internal fixation of pelvic fractures and percutaneous vertebroplasty. CONCLUSION: Our spatially aware approach to transmission image visualization effectively unites patient anatomy with X-ray images by enabling spatial image manipulation that abides image formation. Our proof-of-principle findings indicate potential applications for surgical tasks that mostly rely on orientational information such as placing the acetabular component in total hip arthroplasty, making us confident that the proposed augmented reality concept can pave the way for improving surgical performance and visuo-motor coordination in fluoroscopy-guided surgery.