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De novo design methods hold the promise of reducing the time and cost of antibody discovery, while enabling the facile and precise targeting of predetermined epitopes. Here we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterisation showed that all designs are highly stable, and bind their intended targets with affinities in the nanomolar range without any in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as our method yields similar predictions when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to pre-selected epitopes.summaryA combinatorial method can rapidly design nanobodies for predetermined epitopes, which bind with KDs in the nanomolar range.

Chemical exchange saturation transfer (CEST) enhances solution-state NMR signals of labile and otherwise invisible chemical sites, by indirectly detecting their signatures as a highly magnified saturation of an abundant resonance─for instance, the 1H resonance of water. Stimulated by this sensitivity magnification, this study presents PROgressive Saturation of the Proton Reservoir (PROSPR), a method for enhancing the NMR sensitivity of dilute heteronuclei in static solids. PROSPR aims at using these heteronuclei to progressively deplete the abundant 1H polarization found in most organic and several inorganic solids, and implements this 1H signal depletion in a manner that reflects the spectral intensities of the heteronuclei as a function of their chemical shifts or quadrupolar offsets. To achieve this, PROSPR uses a looped cross-polarization scheme that repeatedly depletes 1H-1H local dipolar order and then relays this saturation throughout the full 1H reservoir via spin-diffusion processes that act as analogues of chemical exchanges in the CEST experiment. Repeating this cross-polarization/spin-diffusion procedure multiple times results in an effective magnification of each heteronucleus's response that, when repeated in a frequency-stepped fashion, indirectly maps their NMR spectrum as sizable attenuations of the abundant 1H NMR signal. Experimental PROSPR examples demonstrate that, in this fashion, faithful wideline NMR spectra can be obtained. These 1H-detected heteronuclear NMR spectra can have their sensitivity enhanced by orders of magnitude in comparison to optimized direct-detect experiments targeting unreceptive nuclei at low natural abundance, using modest hardware requirements and conventional NMR equipment at room temperature.

Aberrant activation of Wnt/β-catenin signaling is strongly associated with many diseases including cancer invasion and metastasis. Small-molecule targeting of the central signaling node of this pathway, β-catenin, is a biologically rational approach to abolish hyperactivation of β-catenin signaling but has been demonstrated to be a difficult task. Herein, we report a drug-like small molecule, ZW4864, that binds with β-catenin and selectively disrupts the protein–protein interaction (PPI) between B-cell lymphoma 9 (BCL9) and β-catenin while sparing the β-catenin/E-cadherin PPI. ZW4864 dose-dependently suppresses β-catenin signaling activation, downregulates oncogenic β-catenin target genes, and abrogates invasiveness of β-catenin-dependent cancer cells. More importantly, ZW4864 shows good pharmacokinetic properties and effectively suppresses β-catenin target gene expression in the patient-derived xenograft mouse model. This study offers a selective chemical probe to explore β-catenin-related biology and a drug-like small-molecule β-catenin/BCL9 disruptor for future drug development.

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level., The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here, the authors employ an approach that uses cryo-EM single particle analysis to track the number and distribution of nucleotides bound to each subunit in the homo-oligomeric MmCpn archaeal chaperonin complex and observe that ATP binds in a statistically random manner to MmCpn both within a ring and across the rings, which shows that there is no cooperativity in ATP binding to archaeal group II chaperonins under the conditions used in this study.

PURPOSE Deuterium metabolic imaging (DMI) maps the uptake of deuterated precursors and their conversion into lactate and other markers of tumor metabolism. Even after leveraging 2 H's short T1 s, DMI's signal-to-noise ratio (SNR) is limited. We hypothesize that a multi-echo balanced steady-state free precession (ME-bSSFP) approach would increase SNR compared to chemical shift imaging (CSI), while achieving spectral isolation of the metabolic precursors and products. METHODS Suitably tuned 2 H ME-bSSFP (five echo times [TEs], ΔTE = 2.2 ms, repetition time [TR]/flip-angle = 12 ms/60°) was implemented at 15.2T and compared to CSI (TR/flip-angle = 95 ms/90°) regarding SNR and spectral isolation, in simulations, in deuterated phantoms and for the in vivo diagnosis of a mouse tumor model of pancreatic adenocarcinoma (N = 10). RESULTS Simulations predicted an SNR increase vs. CSI of 3-5, and that the peaks of 2 H-water, 2 H6,6' -glucose, and 2 H3,3' -lactate can be well isolated by ME-bSSFP; phantoms confirmed this. In vivo, at equal spatial resolution (1.25 × 1.25 mm2 ) and scan time (10 min), 2 H6,6' -glucose's and 2 H3,3' -lactate's SNR were indeed higher for bSSFP than for CSI, three-fold for glucose (57 ± 30 vs. 19 ± 11, P < .001), doubled for water (13 ± 5 vs. 7 ± 3, P = .005). The time courses and overall localization of all metabolites agreed well, comparing CSI against ME-bSSFP. However, a clearer localization of glucose in kidneys and bladder, the detection of glucose-avid rims in certain tumors, and a heterogeneous pattern of intra-tumor lactate production could only be observed using ME-bSSFP's higher resolution. CONCLUSIONS ME-bSSFP provides greater SNR per unit time than CSI, providing for higher spatial resolution mapping of glucose uptake and lactate production in tumors.

Glycan structures are often stabilized by a repertoire of hydrogen-bonded donor/acceptor groups, revealing longer-lived structures that could represent biologically relevant conformations. NMR provides unique data on these hydrogen-bonded networks from multidimensional experiments detecting cross-peaks resulting from through-bond (TOCSY) or through-space (NOESY) interactions. However, fast OH/H2O exchange, and the spectral proximity among these NMR resonances, hamper the use of glycans’ labile protons in such analyses; consequently, studies are often restricted to aprotic solvents or supercooled aqueous solutions. These nonphysiological conditions may lead to unrepresentative structures or to probing a small subset of accessible conformations that may miss “active” glycan conformations. Looped, projected spectroscopy (L-PROSY) has been recently shown to substantially enhance protein NOESY and TOCSY cross-peaks, for 1Hs that undergo fast exchange with water. This study shows that even larger enhancements can be obtained for rapidly exchanging OHs in saccharides, leading to the retrieval of previously undetectable 2D TOCSY/NOESY cross-peaks with nonlabile protons. After demonstrating ≥300% signal enhancements on model monosaccharides, these experiments were applied at 1 GHz to elucidate the structural network adopted by a sialic acid homotetramer, used as a model for α,2–8 linked polysaccharides. High-field L-PROSY NMR enabled these studies at higher temperatures and provided insight previously unavailable from lower-field NMR investigations on supercooled samples, involving mostly nonlabile nuclei. Using L-PROSY’s NOEs and other restraints, a revised structural model for the homotetramer was obtained combining rigid motifs and flexible segments, that is well represented by conformations derived from 40 μs molecular dynamics simulations.

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

The current research reports for the first time the use of blends of poly(e-caprolactone) (PCL) and poly(ester amide) (PEA) for the fabrication of 3D additive manufactured scaffolds. Tailor made PEA was synthesized to afford fully miscible blends of PCL and PEA using different percentages (5, 10, 15 and 20% w/w). Stability, characteristic temperatures and material's compatibility were studied through thermal analyses (i.e., TGA, DSC). Even though DMTA and static compression tests demonstrated the possibility to improve the storage modulus, Young's modulus and maximum stress by increasing the amount of PEA, a decrease of hardness was found beyond a threshold concentration of PEA as the lowest values were achieved for PCL/PEA (20% w/w) scaffolds (from 0.39 ± 0.03 GPa to 0.21 ± 0.02 GPa in the analysed load range). The scaffolds presented a controlled morphology and a fully interconnected network of internal channels. The water contact angle measurements showed a clear increase of hydrophilicity resulting from the addition of PEA. This result was further corroborated with the improved adhesion and proliferation of human mesenchymal stem cells (hMSCs). The presence of PEA also influenced the cell morphology. Better cell spreading and a much higher and homogenous number of cells were observed for PCL/PEA scaffolds when compared to PCL ones.

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.

Histones are typically located within the intracellular compartment, and more specifically, within the nucleus. When histones are located within the extracellular compartment, they change roles and become damage-associated molecular patterns (DAMPs), promoting inflammation and coagulation. Patients with sepsis have increased levels of extracellular histones, which have been shown to correlate with poor prognosis and the development of sepsis-related sequelae, such as end-organ damage. Until now, neutrophils were assumed to be the primary source of circulating histones during sepsis. In this paper, we show that megakaryocytes contain extranuclear histones and transfer histones to their platelet progeny. Upon examination of isolated platelets from patients with sepsis, we identified that patients with sepsis have increased amounts of platelet-associated histones (PAHs), which appear to be correlated with the type of infection. Taken together, these results suggest that megakaryocytes and platelets may be a source of circulating histones during sepsis and should be further explored.

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1 H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW’s enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15 N- 1 H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSR apt ) of the xpt-pbuX operon in Bacillus subtilis . Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1 H NMR spectra allowed us to monitor in real time the changes that GSR apt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1 H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSR apt , is briefly discussed.

International audience; Netrin-1, a secreted protein recently characterized as a relevant cancer therapeutic target, is the antiapoptotic ligand of the dependence receptors deleted in colorectal carcinoma and members of the UNC5H family. Netrin-1 is overexpressed in several aggressive cancers where it promotes cancer progression by inhibiting cell death induced by its receptors. Interference of its binding to its receptors has been shown, through the development of a monoclonal neutralizing antinetrin-1 antibody (currently in phase II of clinical trial), to actively induce apoptosis and tumor growth inhibition. The transcription factor p53 was shown to positively regulate netrin-1 gene expression. We show here that netrin-1 could be a target gene of the N-terminal p53 isoform Δ40p53, independent of full-length p53 activity. Using stable cell lines, harboring wild-type or null-p53, in which Δ40p53 expression could be finely tuned, we prove that Δ40p53 binds to and activates the netrin-1 promoter. In addition, we show that forcing immortalized human skeletal myoblasts to produce the Δ40p53 isoform, instead of full-length p53, leads to the up-regulation of netrin-1 and its receptor UNC5B and promotes cell survival. Indeed, we demonstrate that netrin-1 interference, in the presence of Δ40p53, triggers apoptosis in cancer and primary cells, leading to tumor growth inhibition in preclinical in vivo models. Finally, we show a positive correlation between netrin-1 and Δ40p53 gene expression in human melanoma and colorectal cancer biopsies. Hence, we propose that inhibition of netrin-1 binding to its receptors should be a promising therapeutic strategy in human tumors expressing high levels of Δ40p53.

Huntington's disease is a neurodegenerative disorder resulting from an expanded polyglutamine (polyQ) repeat of the Huntingtin (Htt) protein. Affected tissues often contain aggregates of the N-terminal Htt exon 1 (Htt-Ex1) fragment. The N-terminal N17 domain proximal to the polyQ tract is key to enhance aggregation and modulate Htt toxicity. Htt-Ex1 is intrinsically disordered, yet it has been postulated that under physiological conditions membranes induce the N17 to adopt an α-helical structure, which then plays a key role in regulating Htt protein aggregation. The present study leverages the recently available assignment of NMR peaks in an N17Q17 construct, in order to provide a look into the changes occurring in vitro upon exposing this fragment to various brain extract fragments as well as to synthetic bilayers. Residue-specific changes were observed by 3D HNCO NMR, whose nature was further clarified with ancillary CD and aggregation studies, as well as with molecular dynamic calculations. From this combination of measurements and computations, a unified picture emerges, whereby transient structures consisting of α-helices spanning a fraction of the N17 residues form during N17Q17-membrane interactions. These interactions are fairly dynamic, but they qualitatively mimic more rigid variants that have been discussed in the literature. The nature of these interactions and their potential influence on the aggregation process of these kinds of constructs under physiological conditions are briefly assessed.

We show that TOCSY and multiple-quantum (MQ) 2D NMR spectra can be obtained for mixtures of substrates hyperpolarised by dissolution dynamic nuclear polarisation (D-DNP). This is achieved by combining optimised transfer settings for D-DNP, with ultrafast 2D NMR experiments based on spatiotemporal encoding. TOCSY and MQ experiments are particularly well suited for mixture analysis, and this approach opens the way to significant sensitivity gains for analytical applications of NMR, such as authentication and metabolomics., Using a fast-injection system for dissolution dynamic nuclear polarisation, 1H–1H 2D NMR spectra tailored for the analysis of mixtures are obtained in a single-scan with enhanced sensitivity.

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags., Galaz-Montoya et al. report nanometer-resolution 3D cryo-electron tomography structures of mutant huntingtin (mHTT) and polyglutamine-only (polyQ) filaments in large aggregates free of stains, fixatives, tags, or dehydration artifacts. These results provide a framework for future structural studies of mHTT and polyQ aggregates, thereby improving our understanding of polyQ disorders such as Huntington disease.

Detecting and mapping metabolism in tissues represents a major step in detecting, characterizing, treating and understanding cancers. Recently introduced deuterium metabolic imaging techniques could offer a noninvasive route for the metabolic imaging of animals and humans, based on using 2 H magnetic resonance spectroscopic imaging (MRSI) to detect the uptake of deuterated glucose and the fate of its metabolic products. In this study, 2 H6,6' -glucose was administered to mice cohorts that had been orthotopically implanted with two different models of pancreatic ductal adenocarcinoma (PDAC), involving PAN-02 and KPC cell lines. As the tumors grew, 2 H6,6' -glucose was administered as bolii into the animals' tail veins, and 2 H MRSI images were recorded at 15.2 T. 2D phase-encoded chemical shift imaging experiments could detect a signal from this deuterated glucose immediately after the bolus injection for both the PDAC models, reaching a maximum in the animals' tumors ~ 20 min following administration, and nearly total decay after ~ 40 min. The main metabolic reporter of the cancers was the 2 H3,3' -lactate signal, which MRSI could detect and localize on the tumors when these were 5 mm or more in diameter. Lactate production time traces varied slightly with the animal and tumor model, but in general lactate peaked at times of 60 min or longer following injection, reaching concentrations that were ~ 10-fold lower than those of the initial glucose injection. This 2 H3,3' -lactate signal was only visible inside the tumors. 2 H-water could also be detected as deuterated glucose's metabolic product, increasing throughout the entire time course of the experiment from its ≈10 mM natural abundance background. This water resonance could be imaged throughout the entire abdomen of the animals, including an enhanced presence in the tumor, but also in other organs like the kidney and bladder. These results suggest that deuterium MRSI may serve as a robust, minimally invasive tool for the monitoring of metabolic activity in pancreatic tumors, capable of undergoing clinical translation and supporting decisions concerning treatment strategies. Comparisons with in vivo metabolic MRI experiments that have been carried out in other animal models are presented and their differences/similarities are discussed.

Recent magnetic resonance studies in healthy and cancerous organs have concluded that deuterated metabolites possess highly desirable properties for mapping non-invasively and, as they happen, characterizing glycolysis and other biochemical processes in animals and humans. A promising avenue of this deuterium metabolic imaging (DMI) approach involves looking at the fate of externally administered 2H6,6′-glucose, as it is taken up and metabolized into different products as a function of time. This study employs deuterium magnetic resonance to follow the metabolism of wildtype and preeclamptic pregnant mice models, focusing on maternal and fetoplacental organs over ≈2 h post-injection. 2H6,6′-glucose uptake was observed in the placenta and in specific downstream organs such as the fetal heart and liver. Main metabolic products included 2H3,3′-lactate and 2H-water, which were produced in individual fetoplacental organs with distinct time traces. Glucose uptake in the organs of most preeclamptic animals appeared more elevated than in the control mice (p = 0.02), also higher was the production of 2H-water arising from this glucose. However, the most notable differences arose in the 2H3,3′-lactate concentration, which was ca. two-fold more abundant in the placenta (p = 0.005) and in the fetal (p = 0.01) organs of preeclamptic-like animals, than in control mice. This is consistent with literature reports about hypoxic conditions arising in preeclamptic and growth-restricted pregnancies, which could lead to an enhancement in anaerobic glycolysis. Overall, the present measurements suggest that DMI, a minimally invasive approach, may offer new ways of studying and characterizing health and disease in mammalian pregnancies, including humans.

We investigate thin film resistive thermometry based on metal-to-insulator-transition (niobium nitride) materials down to very low temperature. The variation of the NbN thermometer resistance have been calibrated versus temperature and magnetic field. High sensitivity in tempertaure variation detection is demonstrated through efficient temperature coefficient of resistance. The nitrogen content of the niobium nitride thin films can be tuned to adjust the optimal working temperature range. In the present experiment, we show the versatility of the NbN thin film technology through applications in very different low temperature use-cases. We demonstrate that thin film resistive thermometry can be extended to temperatures below 30 mK with low electrical impedance., Comment: Article accepted for publication in JLTP

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.

Generating a functional proteome requires the ribosome to carefully regulate disparate co-translational processes that determine the fate of nascent polypeptides. With protein synthesis being energetically expensive, the ribosome must balance the costs of efficiently making a protein with those of properly folding it. Emerging as a primary means of regulating this trade-off is the nonuniform rate of translation elongation that defines translation kinetics. The varying speeds with which the ribosome progresses along a transcript have been implicated in several aspects of protein biogenesis, including co-translational protein folding and translational fidelity, as well as gene expression by mediating mRNA decay and protein quality control pathways. The optimal translation kinetics required to efficiently execute these processes can be distinct. Thus, the ribosome is tasked with tightly regulating translation kinetics to balance these processes while maintaining adaptability for changing cellular conditions. In this review, we first discuss the regulatory role of translation elongation in protein biogenesis and what factors influence elongation kinetics. We then describe how changes in translation kinetics signal downstream pathways that dictate the fate of nascent polypeptides. By regulating these pathways, the kinetics of translation elongation has emerged as a critical tool for driving gene expression and maintaining proteostasis through varied mechanisms, including nascent chain folding and binding different ribosome-associated machinery. Indeed, a growing number of examples demonstrate the important role of local changes in elongation kinetics in modulating the pathophysiology of human disease.

While 13C-based Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) experiments offer an attractive alternative for establishing molecular structures, they suffer from low sensitivities arising from the scarcity of spin pairs present at natural abundance. Herein we demonstrate that dissolution dynamic nuclear polarization (dDNP) provides sufficient sensitivity to acquire 1D 13C INADEQUATE spectra in a single scan and at natural abundance. Moreover, if steps are adopted to endow sub-Hertz precision to these measurements, they allow one to measure carbon-carbon J couplings over both one and multiple bonds for each chemical site. As these JCC-couplings are usually sufficiently distinct to enable univocal pairing of the nuclei involved, essentially the same information as in 2D INADEQUATE can be obtained. The feasibility of the method is demonstrated for a range of compounds, including natural products such as α-pinene, menthol and limonene. Features and extensions of this approach are briefly discussed.

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPRER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The levels of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPRER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that can’t be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

Estrogen receptor beta (ERβ) plays a critical role in granulosa cell (GC) functions. The existence of four human ERβ splice isoforms in the ovary suggests their differential implication in 17β-estradiol (E2) actions on GC apoptosis causing follicular atresia. In this study, we investigated whether E2 can regulate ERβ isoforms expression to fine tune its apoptotic activities in human GC. For this purpose, we measured by RT-qPCR the expression of ERβ isoforms in primary culture of human granulosa cells (hGCs) collected from patients undergoing in vitro fertilization, before and after E2 exposure. Besides, we assessed the potential role of ERβ isoforms on cell growth and apoptosis after their overexpression in a human GC line (HGrC1 cells). We confirmed that ERβ1, ERβ2, ERβ4, and ERβ5 isoform mRNAs were predominant over that of ERα in hGCs, and found that E2 selectively regulates mRNA levels of ERβ4 and ERβ5 isoforms in these cells. In addition, we demonstrated that overexpression of ERβ1 and ERβ4 in HGrC1 cells increased cell apoptosis by 225% while ERβ5 or ERβ2 had no effect. Altogether, our study revealed that E2 may influence GC fate by specifically regulating the relative abundance of ERβ isoforms mRNA to modulate the balance between pro-apoptotic and non-apoptotic ERβ isoforms.

Multidimensional NOESY experiments targeting correlations between exchangeable imino and amino protons provide valuable information about base pairing in nucleic acids. It has been recently shown that the sensitivity of homonuclear correlations involving RNA's labile imino protons can be significantly enhanced, by exploiting the repolarization brought about by solvent exchanges. Homonuclear correlations, however, are of limited spectral resolution, and usually incapable of tackling relatively large homopolymers with repeating structures like RNAs. This study presents a heteronuclear-resolved version of those NOESY experiments, in which magnetization transfers between the aqueous solvent and the nucleic acid protons are controlled by selecting specific chemical shift combinations of a coupled 1H-15N spin pair. This selective control effectively leads to a pseudo-3D version of HSQC-NOESY, but with cross-peaks enhanced by ∼2-5× as compared with conventional 2D NOESY counterparts. The enhanced signal sensitivity as well as access to both 15N-1H and 1H-1H NOESY dimensions can greatly facilitate RNA assignments and secondary structure determinations, as demonstrated here with the analysis of genome fragments derived from the SARS-CoV-2 virus.

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

The dynamic structures of microbial communities emerge from the complex network of interactions between their constituent microbial organisms. Quantitative measurements of these microbial interactions are important for understanding and engineering microbial community structure. Here, we present the development and application of the BioMe plate, a redesigned microplate device in which pairs of wells are separated by porous membranes. BioMe facilitates the measurement of dynamic microbial interactions and integrates easily with standard laboratory equipment. We first applied BioMe to recapitulate recently characterized, natural symbiotic interactions between bacteria isolated from theD. melanogastergut microbiome. Specifically, the BioMe plate allowed us to observe the benefit provided by twoLactobacillistrains to anAcetobacterstrain. We next explored the use of BioMe to gain quantitative insight into the engineered obligate syntrophic interaction between a pair ofE. coliamino acid auxotrophs. We integrated experimental observations with a mechanistic computational model to quantify key parameters associated with this syntrophic interaction, including metabolite secretion and diffusion rates. This model also allowed us to explain the slow growth observed for auxotrophs growing in adjacent wells, by demonstrating that under the relevant range of parameters, local exchange between auxotrophs is essential for efficient growth. The BioMe plate provides a scalable and flexible approach for the study of dynamic microbial interactions.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent‐driven exchanges to enhance NOE correlations between exchangeable and non‐exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross‐peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS‐CoV‐2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts., An approach to enhance the sensitivity of 2D NOESY correlations among labile protons is introduced and exemplified with experiments on RNA fragments derived from the SARS‐CoV‐2 genome and with other samples. The approach leads to experiments that are ca. 10‐fold faster and 3‐fold more sensitive than conventional counterparts. The mechanism of this sensitivity boost is explained, and its strengths and limitations are analyzed.

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5γ) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5γ (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo., Analytical Biochemistry, 624, ISSN:0003-2697, ISSN:1096-0309

Many neurodegenerative diseases are characterized by misfolding and aggregation of an expanded polyglutamine tract (polyQ). Huntington's Disease, caused by expansion of the polyQ tract in exon 1 of the Huntingtin protein (Htt), is associated with aggregation and neuronal toxicity. Despite recent structural progress in understanding the structures of amyloid fibrils, little is known about the solution states of Htt in general, and about molecular details of their transition from soluble to aggregation-prone conformations in particular. This is an important question, given the increasing realization that toxicity may reside in soluble conformers. This study presents an approach that combines NMR with computational methods to elucidate the structural conformations of Htt Exon 1 in solution. Of particular focus was Htt's N17 domain sited N-terminal to the polyQ tract, which is key to enhancing aggregation and modulate Htt toxicity. Such in-depth structural study of Htt presents a number of unique challenges: the long homopolymeric polyQ tract contains nearly identical residues, exon 1 displays a high degree of conformational flexibility leading to a scaling of the NMR chemical shift dispersion, and a large portion of the backbone amide groups are solvent-exposed leading to fast hydrogen exchange and causing extensive line broadening. To deal with these problems, NMR assignment was achieved on a minimal Htt exon 1, comprising the N17 domain, a polyQ tract of 17 glutamines, and a short hexameric polyProline region that does not contribute to the spectrum. A pH titration method enhanced this polypeptide's solubility and, with the aid of ≤5D NMR, permitted the full assignment of N17 and the entire polyQ tract. Structural predictions were then derived using the experimental chemical shifts of the Htt peptide at low and neutral pH, together with various different computational approaches. All these methods concurred in indicating that low-pH protonation stabilizes a soluble conformation where a helical region of N17 propagates into the polyQ region, while at neutral pH both N17 and the polyQ become largely unstructured-thereby suggesting a mechanism for how N17 regulates Htt aggregation.

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis., This work was financially supported by NIH grants to R.A. and J.F., and a NIH/NIAID Postdoctoral Fellowship to A.A. S.P. holds the Canada Research Chair in Computational Systems Biology and was supported through computing resources managed by Calcul Québec and Compute Canada. R.G. holds the Ramon y Cajal fellowship from the Spanish Ministry of Economy and Competitiveness (RYC-2015-17517).

This study explored the usefulness of multiple quantitative MRI approaches to detect pancreatic ductal adenocarcinomas in two murine models, PAN-02 and KPC. Methods assayed included 1 H T1 and T2 measurements, quantitative diffusivity mapping, magnetization transfer (MT) 1 H MRI throughout the abdomen and hyperpolarized 13 C spectroscopic imaging. The progress of the disease was followed as a function of its development; studies were also conducted for wildtype control mice and for mice with induced mild acute pancreatitis. Customized methods developed for scanning the motion- and artifact-prone mice abdomens allowed us to obtain quality 1 H images for these targeted regions. Contrasts between tumors and surrounding tissues, however, were significantly different. Anatomical images, T2 maps and MT did not yield significant contrast unless tumors were large. By contrast, tumors showed statistically lower diffusivities than their surroundings (≈8.3 ± 0.4 x 10-4 for PAN-02 and ≈10.2 ± 0.6 x 10-4 for KPC vs 13 ± 1 x 10-3 mm2 s-1 for surroundings), longer T1 relaxation times (≈1.44 ± 0.05 for PAN-02 and ≈1.45 ± 0.05 for KPC vs 0.95 ± 0.10 seconds for surroundings) and significantly higher lactate/pyruvate ratios by hyperpolarized 13 C MR (0.53 ± 0.2 for PAN-02 and 0.78 ± 0.2 for KPC vs 0.11 ± 0.04 for control and 0.31 ± 0.04 for pancreatitis-bearing mice). Although the latter could also distinguish early-stage tumors from healthy animal controls, their response was similar to that in our pancreatitis model. Still, this ambiguity could be lifted using the 1 H-based reporters. If confirmed for other kinds of pancreatic tumors this means that these approaches, combined, can provide a route to an early detection of pancreatic cancer.

SARS-CoV-2 infection (COVID-19) results in local and systemic activation of inflammation and coagulation. In this review article, we will discuss the potential role of coagulation factor Xa (FXa) in the pathophysiology of COVID-19. FXa, a serine protease, has been shown to play a role in the cleavage of SARS-CoV-1 spike protein (SP), with the inhibition of FXa resulting in the inhibition of viral infectivity. FX is known to be primarily produced in the liver, but it is also expressed by multiple cells types, including alveolar epithelium, cardiac myocytes, and macrophages. Considering that patients with preexisting conditions, including cardiopulmonary disease, are at an increased risk of severe COVID-19, we discuss the potential role of increased levels of FX in these patients, resulting in a potential increased propensity to have a higher infectious rate and viral load, increased activation of coagulation and inflammation, and development of fibrosis. With these observations in mind, we postulate as to the potential therapeutic role of FXa inhibitors as a prophylactic and therapeutic treatment for high-risk patients with COVID-19.

Wolbachia is an intracellular bacterial symbiont of arthropods notorious for inducing many reproductive manipulations that foster its dissemination. Wolbachia affects many aspects of host biology, including metabolism, longevity and physiology, being described as a nutrient provisioning or metabolic parasite, depending on the host-microbe association. Sirtuins (SIRTs) are a family of NAD+-dependent post-translational regulatory enzymes known to affect many of the same processes altered by Wolbachia, including aging and metabolism, among others. Despite a clear overlap in control of host-derived pathways and physiology, no work has demonstrated a link between these two regulators. We used genetically tractable Drosophila melanogaster to explore the role of sirtuins in shaping signaling pathways in the context of a host-symbiont model. By using transcriptional profiling and metabolic assays in the context of genetic knockouts/over-expressions, we examined the effect of several Wolbachia strains on host sirtuin expression across distinct tissues and timepoints. We also quantified the downstream effects of the sirtuin x Wolbachia interaction on host glucose metabolism, and in turn, how it impacted Wolbachia titer. Our results indicate that the presence of Wolbachia is associated with (1) reduced sirt-4 expression in a strain-specific manner, and (2) alterations in host glutamate dehydrogenase expression and ATP levels, key components of glucose metabolism. We detected high glucose levels in Wolbachia-infected flies, which further increased when sirt-4 was over-expressed. However, under sirt-4 knockout, flies displayed a hypoglycemic state not rescued to normal levels in the presence of Wolbachia. Finally, whole body sirt-4 over-expression resulted in reduced Wolbachia ovarian titer. Our results expand knowledge of Wolbachia-host associations in the context of a yet unexplored class of host post-translational regulatory enzymes with implications for conserved host signaling pathways and bacterial titer, factors known to impact host biology and the symbiont’s ability to spread through populations., Author summary Here we utilize Drosophila genetic tools to dissect how Wolbachia interacts with a class of host enzymes known as sirtuins, widely recognized as key regulators of host biological processes including metabolism, longevity, stress response, and others. By focusing on the sirt-4 gene, we demonstrate that the presence of Wolbachia is associated with a consistent reduction in sirt-4 transcriptional levels across multiple tissues and timepoints. The bacterium is also associated with alterations in the expression of glutamate dehydrogenase and total ATP levels, key components of the glucose metabolism pathway. Finally, we show that the Wolbachia-sirt-4 interaction is associated with the modulation of host glucose homeostasis and, that sirt-4 negatively affects the growth of the bacteria in the host reproductive tissues. Our results further expand knowledge of host-microbe interactions in the context of host physiological manipulation by intracellular bacteria, offering new ways to interpret Wolbachia’s successful dissemination in insect populations in nature.

Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group 1H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D 1H–15N correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this “HyperW” method to four proteins exhibiting a gamut of exchange behaviors: PhoA(350–471), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA4(350–471) HyperW sensitivity enhancements were ≥300×, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of ≥100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations—including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized “structural” waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts—are considered to account for them.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform ‘hyperpolarization-selective’ signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN). Electronic supplementary material The online version of this article (10.1007/s10858-020-00301-5) contains supplementary material, which is available to authorized users.

Significance The endoplasmic reticulum (ER) is the site for folding and maturation of secreted and membrane proteins. When the ER protein-folding machinery is overwhelmed, misfolded proteins trigger ER stress, which is frequently linked to human diseases, including cancer and neurodegeneration. Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident sensor that assembles into large clusters of previously unknown organization upon its activation by unfolded peptides. We demonstrate that IRE1 clusters are topologically complex dynamic structures that remain contiguous with the ER membrane throughout their lifetime. The majority of clustered IRE1 molecules are diffusionally trapped inside the clusters until IRE1 signaling attenuates, at which point they are released back into the ER through a pathway that is functionally distinct from cluster assembly., The endoplasmic reticulum (ER) membrane-resident stress sensor inositol-requiring enzyme 1 (IRE1) governs the most evolutionarily conserved branch of the unfolded protein response. Upon sensing an accumulation of unfolded proteins in the ER lumen, IRE1 activates its cytoplasmic kinase and ribonuclease domains to transduce the signal. IRE1 activity correlates with its assembly into large clusters, yet the biophysical characteristics of IRE1 clusters remain poorly characterized. We combined superresolution microscopy, single-particle tracking, fluorescence recovery, and photoconversion to examine IRE1 clustering quantitatively in living human and mouse cells. Our results revealed that: 1) In contrast to qualitative impressions gleaned from microscopic images, IRE1 clusters comprise only a small fraction (∼5%) of the total IRE1 in the cell; 2) IRE1 clusters have complex topologies that display features of higher-order organization; 3) IRE1 clusters contain a diffusionally constrained core, indicating that they are not phase-separated liquid condensates; 4) IRE1 molecules in clusters remain diffusionally accessible to the free pool of IRE1 molecules in the general ER network; 5) when IRE1 clusters disappear at later time points of ER stress as IRE1 signaling attenuates, their constituent molecules are released back into the ER network and not degraded; 6) IRE1 cluster assembly and disassembly are mechanistically distinct; and 7) IRE1 clusters’ mobility is nearly independent of cluster size. Taken together, these insights define the clusters as dynamic assemblies with unique properties. The analysis tools developed for this study will be widely applicable to investigations of clustering behaviors in other signaling proteins.

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time. We demonstrate that misfolded or partially folded disulfide intermediates are indistinguishable from the unfolded state when focusing on the backbone NMR signals, which provide information on the formation of only the final, native state. However, 13C labeling of cysteine side chains differentiated transient intermediates from the unfolded and native states and reported on disulfide bond formation in real time. The cysteine pairings in a dominant intermediate were identified using 13C-edited three-dimensiona...

Lysosomes keep neuronal stem cells young An important consequence of aging is loss of regenerative capacity in stem cells, particularly those of the nervous system. Leeman et al. isolated quiescent and activated stem cells from mice and compared their transcriptomes. The findings emphasize the role of large lysosomes in quiescent neuronal stem cells in which aggregated proteins accumulate. Treatments that stimulated lysosomal function allowed aged quiescent stem cells to clear protein aggregates and restored the cells' ability to be activated. Such restoration of stem cell function might alleviate compromised proteostasis in aging. Science , this issue p. 1277

The study has revealed the regularities of the effect produced by increasing the content of scale of steel 12Cr18Ni10Ti in the charge from 5 to 75 mass % on the contents of the products of carbon thermal reduction of oxide waste of corrosion-resistant steels. The concentration of Ni is increased from 0.8 to 7.0 mass % when the Cr content ranges from 15.9 to 17.1 mass %. The concentration of Cr in the extraction products within the range of 16.1–17.1 mass% is provided with the content of scale of 95Cr18 steel in the charge in the range from 5 to 55 mass %. It has been found that metallization products mainly consist of a solid solution of alloying elements in α-Fe. Fe 3 O 4 , Fe 3 C, and Fe 2 C were also identified. The microstructure of the extraction products is spongy and disordered. The particles are sintered, with varying Cr and Ni contents in the ranges of 7.47 to 18.03 mass % and 2.97–10.40 mass %, respectively. The study has helped achieve environmentally safe conditions for solid-phase extraction of chrome and nickel containing industrial wastes from the production of corrosion-resistant steels with the return of the alloyed product to the welding industry