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BACKGROUND: Lenalidomide (R), bortezomib (V), and dexamethasone (d) is a standard-of-care regimen in newly diagnosed multiple myeloma (NDMM); however, characteristics and outcomes for nontransplanted patients receiving frontline RVd are not well understood. PATIENTS: The Connectâ MM Registry is a large, US, multicenter, prospective observational cohort study of NDMM patients. METHODS: This analysis investigated characteristics and outcomes of patients who received RVd alone or followed by Rd or R (RVd ± Rd/R) who did not undergo frontline autologous stem cell transplantation. RESULTS: As of August 2021, 314 of 1979 nontransplanted patients received RVd ± Rd/R as initial therapy. Of these, 135 were aged ≤ 65 years and 179 were > 65 years. 108 patients had time to relapse (TTR) of ≤ 12 months and 182 had TTR > 12 months. Baseline characteristics were comparable regardless of TTR and age group except renal function, which was more commonly impaired in older patients. Among patients aged ≤ 65 and > 65 years, median duration of first-line treatment was 6.3 and 9.0 months, median time to next line for those who received second-line therapy was 15.5 and 15.2 months, median progression-free survival (PFS) was 19.3 and 23.0 months, and median overall survival was 60.0 and 59.1 months, respectively. High-risk disease (per IMWG criteria) and high serum calcium were associated with higher hazard of progression or death; the adjusted PFS hazard ratio with respect to age (≤ 65 vs. > 65 years) based on multivariable analysis was 1.18 (0.89-1.57; P = .25). CONCLUSION: These results indicate RVd is active across age groups and provide a better understanding of outcomes with RVd in NDMM.
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The piezoelectric properties of two-dimensional semiconductor nanobubbles present remarkable potential for application in flexible optoelectronic devices, and the piezoelectric field has emerged as an efficacious pathway for both the separation and migration of photogenerated electron-hole pairs, along with inhibition of recombination. However, the comprehension and control of photogenerated carrier dynamics within nanobubbles still remain inadequate. Hence, this study is dedicated to underscore the importance of in situ detection and detailed characterization of photogenerated electron-hole pairs in nanobubbles to enrich understanding and strategic manipulation in two-dimensional semiconductor materials. Utilizing frequency modulation kelvin probe force microscopy (FM-KPFM) and strain gradient distribution techniques, the existence of a piezoelectric field in monolayer WS2 nanobubbles was confirmed. Combining w/o and with illumination FM-KPFM, second-order capacitance gradient technique and in situ nanoscale tip-enhanced photoluminescence characterization techniques, the interrelationships among the piezoelectric effect, interlayer carrier transfer, and the funneling effect for photocarrier dynamics process across various nanobubble sizes were revealed. Notably, for a WS2/graphene bubble height of 15.45 nm, a 0 mV surface potential difference was recorded in the bubble region w/o and with illumination, indicating a mutual offset of piezoelectric effect, interlayer carrier transfer, and the funneling effect. This phenomenon is prevalent in transition metal dichalcogenides materials exhibiting inversion symmetry breaking. The implication of our study is profound for advancing the understanding of the dynamics of photogenerated electron-hole pair in nonuniform strain piezoelectric systems, and offers a reliable framework for the separation and modulation of photogenerated electron-hole pair in flexible optoelectronic devices and photocatalytic applications.
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Photoelectrochemical (PEC) water splitting provides a scalable and integrated platform to harness renewable solar energy for green hydrogen production. The practical implementation of PEC systems hinges on addressing three critical challenges: enhancing energy conversion efficiency, ensuring long-term stability, and achieving economic viability. Metal-insulator-semiconductor (MIS) heterojunction photoelectrodes have gained significant attention over the last decade for their ability to efficiently segregate photogenerated carriers and mitigate corrosion-induced semiconductor degradation. This review discusses the structural composition and interfacial intricacies of MIS photoelectrodes tailored for PEC water splitting. The application of MIS heterostructures across various semiconductor light-absorbing layers, including traditional photovoltaic-grade semiconductors, metal oxides, and emerging materials, is presented first. Subsequently, this review elucidates the reaction mechanisms and respective merits of vacuum and non-vacuum deposition techniques in the fabrication of the insulator layers. In the context of the metal layers, this review extends beyond the conventional scope, not only by introducing metal-based cocatalysts, but also by exploring the latest advancements in molecular and single-atom catalysts integrated within MIS photoelectrodes. Furthermore, a systematic summary of carrier transfer mechanisms and interface design principles of MIS photoelectrodes is presented, which are pivotal for optimizing energy band alignment and enhancing solar-to-chemical conversion efficiency within the PEC system. Finally, this review explores innovative derivative configurations of MIS photoelectrodes, including back-illuminated MIS photoelectrodes, inverted MIS photoelectrodes, tandem MIS photoelectrodes, and monolithically integrated wireless MIS photoelectrodes. These novel architectures address the limitations of traditional MIS structures by effectively coupling different functional modules, minimizing optical and ohmic losses, and mitigating recombination losses.
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2D transition metal dichalcogenides (TMDs) exhibit exceptional resilience to mechanical deformation. Applied strain can have pronounced effects on properties such as the bandgaps and exciton dynamics of TMDs, via deformation potentials and electromechanical coupling. In this work, we use piezoresponse force microscopy to show that the inhomogeneous strain from nanobubbles produces dramatic, localized enhancements of the electromechanical response of monolayer MoS2. Nanobubbles with diameters under 100 nm consistently produce an increased piezoresponse that follows the features' topography, while larger bubbles exhibit a halo-like profile, with maximum piezoresponse near the periphery. We show that spatial filtering enables these effects to be eliminated in the quantitative determination of effective piezoelectric or flexoelectric coefficients. Numerical strain modeling reveals a correlation between the hydrostatic strain gradient and the effective piezoelectric coefficient in large MoS2 nanobubbles, suggesting a localized variation in electromechanical coupling due to symmetry reduction induced by inhomogeneous strain.
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BACKGROUND: Effective primary care necessitates follow-up actions by the patient beyond the visit. Prior research suggests room for improvement in patient adherence. OBJECTIVE: This study sought to understand patients' views on their primary care visits, the plans generated therein, and their self-reported adherence after 3 months. METHODS: As part of a large multisite cluster randomized pragmatic trial in 3 health care organizations, patients completed 2 surveys-the first within 7 days after the index primary care visit and another 3 months later. For this analysis of secondary outcomes, we combined the results across all study participants to understand patient adherence to care plans. We recorded patient characteristics and survey responses. Cross-tabulation and chi-square statistics were used to examine bivariate associations, adjusting for multiple comparisons when appropriate. We used multivariable logistic regression to assess how patients' intention to follow, agreement, and understanding of their plans impacted their plan adherence, allowing for differences in individual characteristics. Qualitative content analysis was conducted to characterize the patient's self-reported plans and reasons for adhering (or not) to the plan 3 months later. RESULTS: Of 2555 patients, most selected the top box option (9=definitely agree) that they felt they had a clear plan (n=2011, 78%), agreed with the plan (n=2049, 80%), and intended to follow the plan (n=2108, 83%) discussed with their provider at the primary care visit. The most common elements of the plans reported included reference to exercise (n=359, 14.1%), testing (laboratory, imaging, etc; n=328, 12.8%), diet (n=296, 11.6%), and initiation or adjustment of medications; (n=284, 11.1%). Patients who strongly agreed that they had a clear plan, agreed with the plan, and intended to follow the plan were all more likely to report plan completion 3 months later (P<.001) than those providing less positive ratings. Patients who reported plans related to following up with the primary care provider (P=.008) to initiate or adjust medications (P≤.001) and to have a specialist visit were more likely to report that they had completely followed the plan (P=.003). Adjusting for demographic variables, patients who indicated intent to follow their plan were more likely to follow-through 3 months later (P<.001). Patients' reasons for completely following the plan were mainly that the plan was clear (n=1114, 69.5%), consistent with what mattered (n=1060, 66.1%), and they were determined to carry through with the plan (n=887, 53.3%). The most common reasons for not following the plan were lack of time (n=217, 22.8%), having decided to try a different approach (n=105, 11%), and the COVID-19 pandemic impacted the plan (n=105, 11%). CONCLUSIONS: Patients' initial assessment of their plan as clear, their agreement with the plan, and their initial willingness to follow the plan were all strongly related to their self-reported completion of the plan 3 months later. Patients whose plans involved lifestyle changes were less likely to report that they had "completely" followed their plan. TRIAL REGISTRATION: ClinicalTrials.gov NCT03385512; https://clinicaltrials.gov/study/NCT03385512. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/30431.
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SUMMARYInfectious bacteria have both intrinsic and acquired mechanisms to combat harmful biocides that enter the cell. Through adaptive pressures, many of these pathogens have become resistant to many, if not all, of the current antibiotics used today to treat these often deadly infections. One prominent mechanism is the upregulation of efflux systems, especially the resistance-nodulation-cell division class of exporters. These tripartite systems consist of an inner membrane transporter coupled with a periplasmic adaptor protein and an outer membrane channel to efficiently transport a diverse array of substrates from inside the cell to the extracellular space. Detailed mechanistic insight into how these inner membrane transporters recognize and shuttle their substrates can ultimately inform both new antibiotic and efflux pump inhibitor design. This review examines the structural basis of substrate recognition of these pumps and the molecular mechanisms underlying multidrug extrusion, which in turn mediate antimicrobial resistance in bacterial pathogens.
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Antibacterianos , Proteínas de Membrana Transportadoras , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/farmacologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Divisão Celular , Farmacorresistência Bacteriana Múltipla , Transporte Biológico , Modelos Moleculares , Farmacorresistência BacterianaRESUMO
Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein-ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.
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Campylobacter jejuni is a foodborne pathogen commonly found in the intestinal tracts of animals. This pathogen is a leading cause of gastroenteritis in humans. Besides its highly infectious nature, C. jejuni is increasingly resistant to a number of clinically administrated antibiotics. As a consequence, the Centers for Disease Control and Prevention has designated antibiotic-resistant Campylobacter as a serious antibiotic resistance threat in the United States. The C. jejuni CosR regulator is essential to the viability of this bacterium and is responsible for regulating the expression of a number of oxidative stress defense enzymes. Importantly, it also modulates the expression of the CmeABC multidrug efflux system, the most predominant and clinically important system in C. jejuni that mediates resistance to multiple antimicrobials. Here, we report structures of apo-CosR and CosR bound with a 21 bp DNA sequence located at the cmeABC promotor region using both single-particle cryo-electron microscopy and X-ray crystallography. These structures allow us to propose a novel mechanism for CosR regulation that involves a long-distance conformational coupling and rearrangement of the secondary structural elements of the regulator to bind target DNA. IMPORTANCE: Campylobacter jejuni has emerged as an antibiotic-resistant threat worldwide. CosR is an essential regulator for this bacterium and is important for Campylobacter adaptation to various stresses. Here, we describe the structural basis of CosR binding to target DNA as determined by cryo-electron microscopy and X-ray crystallography. Since CosR is a potential target for intervention, our studies may facilitate the development of novel therapeutics to combat C. jejuni infection.
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Campylobacter jejuni , Campylobacter , Animais , Humanos , Campylobacter jejuni/genética , Microscopia Crioeletrônica , Campylobacter/genética , Antibacterianos/metabolismo , DNA/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.
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GTP Fosfo-Hidrolases , Dinâmica Mitocondrial , Animais , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mamíferos/metabolismoRESUMO
Strain engineering modifies the optical and electronic properties of atomically thin transition metal dichalcogenides. Highly inhomogeneous strain distributions in two-dimensional materials can be easily realized, enabling control of properties on the nanoscale; however, methods for probing strain on the nanoscale remain challenging. In this work, we characterize inhomogeneously strained monolayer MoS2 via Kelvin probe force microscopy and electrostatic gating, isolating the contributions of strain from other electrostatic effects and enabling the measurement of all components of the two-dimensional strain tensor on length scales less than 100 nm. The combination of these methods is used to calculate the spatial distribution of the electrostatic potential resulting from piezoelectricity, presenting a powerful way to characterize inhomogeneous strain and piezoelectricity that can be extended toward a variety of 2D materials.
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PURPOSE: Response EvaluationCriteriain Solid Tumors (RECIST) is commonly used to assess response to anti-cancer therapies. However, its application after lung stereotactic ablative radiotherapy (SABR) is complicated by radiation-induced lung changes. This study assesses the frequency of progressive disease (PD) by RECIST following lung SABR and correlates this with actual treatment outcomes as determined by longitudinal follow-up. METHODS AND MATERIALS: We reviewed patients treated with lung SABR for primary lung tumors or oligometastases between 2010 and 2015. Patients were treated with SABR doses of 54-60 Gy in 3-8 fractions. All follow-up scans were assessed and the treated lesion was serially measured over time, with the maximum diameter on axial CT slices used for RECIST calculations. Lesions demonstrating PD by RECIST criteria were identified and subsequently followed for long-term outcomes. The final 'gold-standard' assessment of response was based on size changes after PD and, as available, positron emission tomography scan and/or biopsy. RESULTS: Eighty-eight lesions met inclusion criteria. Seventy-five were lung primaries and thirteen were lung metastases. Median follow-up was 52 months (interquartile range: 33-68). Two-thirds (66 %, 58/88) of treated lesions met RECIST criteria for PD; however, local recurrence was only confirmed in 16 % (9/58) of cases. Most lesions that triggered PD by RECIST (47/58, 81 %) were ultimately found not to represent recurrence, while a minority (2/58, 3 %) had an uncertain response. The positive predictive value [PPV] of a RECIST defined PD event was 0.16. If PD was triggered within 12-months post-treatment, PPV was 0.08, compared to 0.21 for lesions triggering PD after 12-months. CONCLUSION: Using RECIST criteria, two-thirds of patients treated with lung SABR met criteria for PD. However, only a minority had recurrence, leading to a poor PPV of RECIST. This highlights the limitations of RECIST in this setting and provides context for physicians when interpreting post-lung SABR imaging.
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Neoplasias Pulmonares , Radiocirurgia , Humanos , Critérios de Avaliação de Resposta em Tumores Sólidos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/patologia , Resultado do Tratamento , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Radiocirurgia/métodos , Pulmão/diagnóstico por imagem , Pulmão/patologiaRESUMO
Multidrug efflux is one of the major mechanisms of antibiotic resistance identified in clinical isolates of the human pathogen Acinetobacter baumannii. The multiple antibiotic resistance in this species is often enabled by the overproduction of the tripartite efflux pump AdeABC. In this pump, AdeB is the inner membrane transporter from the resistance-nodulation-division (RND) superfamily of proteins, which is responsible for the recognition and efflux of multiple structurally unrelated compounds. Like other RND transporters, AdeB is a trimeric protein with ligand-binding sites located in the large periplasmic domains. Previous structural studies, however, highlighted the uniqueness of AdeB interactions with ligands. Up to three ligand molecules were bound to one protomer of AdeB, mapping its substrate translocation path. In this study, we introduced single and double substitutions in the identified ligand-binding sites of AdeB. Our results show that the mechanism of substrate translocation by AdeB is different from that of other characterized RND transporters and that the functional interactions between the sites are nonadditive. We identified AdeB mutants with both the loss and the gain of antibiotic susceptibility phenotypes, as well as AdeB mutations making A. baumannii cells overproducing such pump variants even more susceptible to multiple antibiotics than efflux-deficient cells. IMPORTANCE Multidrug efflux pumps of the resistance-nodulation-division superfamily of proteins are important contributors to various aspects of bacterial physiology and antibiotic resistance. Studies of the best-characterized model transporter AcrB from Escherichia coli suggested that these transporters operate by a functional rotation mechanism in which various substrates bind to at least two different binding sites. This study suggests that the mechanism of AdeB is distinct and that the binding sites in this transporter are functionally linked.
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Acinetobacter baumannii , Proteínas de Escherichia coli , Humanos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Ligantes , Antibacterianos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Sítios de Ligação , Escherichia coli/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
An extremely rapid process for self-assembling well-ordered, nano, and microparticle monolayers via a novel aerosolized method is presented. The novel technique can reach monolayer self-assembly rates as high as 268 cm2 min-1 from a single aerosolizing source and methods to reach faster monolayer self-assembly rates are outlined. A new physical mechanism describing the self-assembly process is presented and new insights enabling high-efficiency nanoparticle monolayer self-assembly are developed. In addition, well-ordered monolayer arrays from particles of various sizes, surface functionality, and materials are fabricated. This new technique enables a 93× increase in monolayer self-assembly rates compared to the current state of the art and has the potential to provide an extremely low-cost option for submicron nanomanufacturing.
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PURPOSE: Radiation therapy (RT) is an important treatment modality for patients with multiple myeloma (MM). Although patients are living longer with MM, they are more likely to have comorbidities related to treatment, such as bone pain; however, RT can provide symptom relief. To date, the characterization of patients who have received RT in the real-world setting has been limited. METHODS AND MATERIALS: The Connect® MM Registry is a large, US multicenter, prospective observational cohort study of adult patients with newly diagnosed MM from mostly community sites. RT utilization and outcomes were analyzed quarterly throughout treatment. Factors associated with RT use were identified via multivariable analysis. RESULTS: A total of 3011 patients were enrolled in the Connect MM Registry with 903 patients (30%) having received RT at any time. There was a significant difference (P < .05) in overall RT use among patients with an Eastern Cooperative Oncology Group performance status of 0 to 1 versus ≥2, International Staging System disease stage I/II versus III, a history of plasmacytoma or a novel agent in their first regimen, and any number of bone lesions or severe osteoporosis/fracture. RT use was associated with having bone lesions or severe osteoporosis (vs not having bone lesions). Additionally, RT use was associated with ethnicity (Hispanic vs not) and Connect MM Registry cohort (cohort 1 [enrolled 2009-2011] vs 2 [enrolled 2012-2016]). In the 6 months before death, increased RT use was associated with increasing number of treatment lines (P < .0001) and high- versus standard-risk disease (per International Myeloma Working Group criteria; P = .0028). CONCLUSIONS: Real-world results from the Connect MM Registry show RT is frequently used and is associated with clinical factors, including performance status and disease stage. Earlier in MM diagnosis, RT may be used as an adjunct to palliate symptoms or delay systemic therapy. Toward the end of life, RT is more frequently used for palliation when treatment options are often limited.
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Mieloma Múltiplo , Osteoporose , Adulto , Humanos , Mieloma Múltiplo/radioterapia , Estudos Prospectivos , Etnicidade , Sistema de RegistrosRESUMO
New approaches for combating microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen, Salmonella enterica serotype Typhimurium (S. Typhimurium), in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy. Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome, acrAB is required for growth, the EPM analogs are bacteriostatic, and the EPM analogs increase the potency of antibiotics. These data suggest that under macrophage-like conditions, the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics.IMPORTANCEBacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics.
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New approaches for combatting microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division (RND) family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen, Salmonella enterica serotype Typhimurium (S. Typhimurium) in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy (cryo-EM). Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome, acrAB is required for growth, the EPM analogs are bacteriostatic, and increase the potency of antibiotics. These data suggest that under macrophage-like conditions the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics.
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The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simultaneously identify and solve high-resolution structures of nine essential mitochondrial enzymes with key metabolic functions, including fatty acid catabolism, reactive oxidative species clearance, and amino acid metabolism. Our methodology also identified multiple distinct members of the acyl-CoA dehydrogenase family. This work highlights the potential of cryo-EM to explore tissue proteomics at the atomic level.
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Mitocôndrias , Proteômica , Humanos , Mitocôndrias/metabolismo , Fígado/metabolismo , OxirreduçãoRESUMO
The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.
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Olho , Vitamina A , Animais , Camundongos , Proteínas Celulares de Ligação ao Retinol/metabolismo , Ligantes , Vitamina A/metabolismo , Proteínas de TransporteRESUMO
Moseng et al. recently reported four cryo-electron microscopy structures of the human Na-K-2Cl cotransporter-1 (hNKCC1), both in the absence and presence of bound loop diuretic (furosemide or bumetanide). This research article included high-resolution structural information for a previously undefined structure of apo-hNKCC1 containing both the transmembrane and cytosolic carboxyl-terminal domains. The manuscript also demonstrated various conformational states of this cotransporter induced by diuretic drugs. On the basis of the structural information, the authors proposed a scissor-like inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1. This work has provided important insights into the mechanism of inhibition and substantiated the concept of a long-distance coupling involving movements of both the transmembrane and carboxyl-terminal cytoplasmic domains for inhibition.
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Bumetanida , Furosemida , Humanos , Microscopia Crioeletrônica , Bumetanida/farmacologia , Citosol , Conformação MolecularRESUMO
Genomic data variability from laboratory reports can impact clinical decisions and population-level analyses; however, the extent of this variability and the impact on the data's value are not well characterized. This pilot study used anonymized genetic and genomic test reports from the Connect Myeloid Disease Registry (NCT01688011), a multicenter, prospective, observational cohort study of patients with newly diagnosed myelodysplastic syndromes, acute myeloid leukemia, or idiopathic cytopenia of undetermined significance, to analyze laboratory test variabilities and limitations. Results for 56 randomly selected patients enrolled in the Registry were independently extracted and evaluated (data cutoff, January 2020). Ninety-five reports describing 113 assay results from these 56 patients were analyzed for discrepancies. Almost all assay results [101 (89%)] identified the sequencing technology applied, and 94 (83%) described the test limitations; 95 (84%) described the limits of detection, but none described the limit of blank for detecting false positives. RNA transcript identifiers were not provided for 20 (43%) variants analyzed by next-generation sequencing and reported by the same laboratory. Of 42 variants with variant allele frequencies ≥30%, 16 (38%) of the variants did not have report text indicating that the variants might be germline. Variabilities and lack of standardization present challenges for incorporating this information into clinical care and render data collation ineffective and unreliable for large-scale use in centralized databases for therapeutic discovery.