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Hyperpolarization by dissolution dynamic nuclear polarization has brought highly sensitive magnetic resonance to reality but there still remains severe limitations. Here the authors show an approach relying on the generation of hyperpolarizing polymers that bear a dual function.

The low sensitivity of conventional nuclear magnetic resonance experiments can be overcome, in suitable cases, by employing hyperpolarization methodologies. One such technique, dissolution-dynamic nuclear polarization, provides a robust means of strongly polarizing a variety of small molecules. A drawback of the dissolution-dynamic nuclear polarization approach, the excessively long polarization timescales for insensitive nuclei, has been circumvented by using cross-polarization radiofrequency pulse sequences, which in general yield quick and substantial 13C polarizations. However, the capacity to effectively perform efficient cross-polarization experiments under dissolution-dynamic nuclear polarization conditions remains challenging, and polarization quantification can be plagued by additional complications including spurious background signals from the probe. Here we propose a background-free 1H-13C radiofrequency coil specifically designed for use in cross-polarization experiments on samples of up to 500 μL in volume at liquid helium temperatures. We additionally introduce simple guidelines for the optimization and implementation of cross-polarization radiofrequency pulse sequences. Experimental demonstrations of 13C polarizations reaching ∼60% are presented for the case of [1-13C]sodium acetate.

Dissolution-dynamic nuclear polarization can be boosted by employing multiple-contact cross-polarization techniques to transfer polarization from 1H to 13C spins. The method is efficient and significantly reduces polarization build-up times, however, it involves high-power radiofrequency pulses in a superfluid helium environment which limit its implementation and applicability and prevent a significant scaling-up of the sample size. We propose to overcome this limitation by a stepwise transfer of polarization using a low-energy and low-peak power radiofrequency pulse sequence where the 1H→13C polarization transfer is mediated by a dipolar spin order reservoir. An experimental demonstration is presented for [1-13C]sodium acetate. A solid-state 13C polarization of ∼43.5% was achieved using this method with a build-up time constant of ∼5.1 minutes, leading to a ∼27.5% 13C polarization in the liquid-state after sample dissolution. The low-power multiple-step polarization transfer efficiency achieved with respect to the most advanced and highest-power multiple-contact cross-polarization approach was found to be ∼0.69.

Dissolution dynamic nuclear polarization (dDNP) has become a hyperpolarization method of choice for enhancing nuclear magnetic resonance (NMR) signals. Nuclear spins are polarized in solid frozen samples (in a so-called polarizer) that are subsequently dissolved and transferred to an NMR spectrometer for high sensitivity detection. One of the critical challenges of dDNP is that it requires both a fast transfer to limit nuclear spin relaxation losses as well as stability to guarantee high resolution (no bubbles nor turbulences). Here we describe the design, construction and performances of such a transfer and injection system, that features a 5 m/s speed and sub-Hz spectral resolution upon arrival at the detection spot. We demonstrate the use of such a system for inter-magnet distances of up to 10 m.

Background: In sub-Saharan Africa, rural areas have lower rates of access to safe drinking water compared to urban areas. We investigated predictors of Escherichia coli contamination in drinking water of rural households in Ghana, Malawi, Mozambique, Niger, Rwanda, Uganda, and Zambia., Methods: We used a population-based, cluster randomized sampling design to select rural households in each country. Household interviews on water access, sanitation, and hygiene (WaSH) practices and demographic characteristics were conducted and water samples from every fifth household were collected and enumerated for E. coli. Negative binomial regression models with survey sampling weights were run to evaluate predictors of E. coli contamination., Results: A total of 18,747 rural household surveys (2,378-2,804 per country) were conducted and a total of 3,848 water samples (460-660 per country) were collected. Of surveyed rural households, 61-78% of households had high (11-100 E. coli cfu/100 mL) or very high (>100 cfu/100 mL) risk water quality in Ghana, Niger, and Uganda. Statistically significant WaSH predictors associated with lower E. coli incidence rates included using an improved-type primary water source (Mozambique), storing water in a narrow-mouthed container or container with a spigot (Niger), having continuous water supply during the dry season (Ghana), paying for water service (Rwanda), having soap or ash at handwashing points (Mozambique), having an improved-type household sanitation facility (Malawi), and attaining an education level greater than primary school (Niger and Zambia)., Conclusion: This study highlights the variability in WaSH access between rural areas of the study countries in association with microbial drinking water quality., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Georgia Kayser reports financial support was provided by National Institute of Environmental Health Sciences. Pete Kolsky (listed in acknowledgments) reports financial support was provided by World Vision International. Co-author currently serving on the editorial board for the International Journal of Hygiene and Environmental Health - Jamie Bartram If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Hepatitis B virus (HBV) antiviral administration and adherence are essential to reach the World Health Organization's 2030 hepatitis elimination goals. As HBV treatment guidelines are now simplified and expanded, adherence to treatment will be critical, but challenges to adherence are poorly studied. After introducing tenofovir disoproxil fumarate (TDF) monotherapy to expectant mothers with high-risk HBV in Kinshasa, DRC, we conducted semi-structured interviews to understand medication adherence behaviors, to complement pill counts and measurement of TDF metabolite levels. Key facilitators of adherence identified included trust in healthcare workers, a better understanding of HBV, and family support. Identified barriers included fear of stigma and low health literacy. Knowledge about HBV disease remains low, underscoring the importance of ongoing education of clinic staff and patients alike., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objective: Despite global reductions in hepatitis B virus (HBV) prevalence, an estimated 6.2 million children are infected, two-thirds of whom live in the WHO Africa region. We sought to characterize childhood HBV to inform elimination efforts in the Democratic Republic of Congo (DRC), one of the largest and most populous African countries., Methods: Using the most recent (2013-14) nationally representative Demographic and Health Survey in the DRC, we analyzed HBV surface antigen (HBsAg) on dried blood spots and associated survey data from children aged 6-59 months. We estimated HBsAg-positivity prevalence nationally, regionally, and by potential correlates of infection. We evaluated spatial variation in HBsAg-positivity prevalence, overall and by age, sex, and vaccination status., Findings: Using data from 5,679 children, we found national HBsAg-positivity prevalence was 1.3% (95% CI: 0.9%-1.7%), but ranged from 0.0% in DRC's capital city province, Kinshasa, to 5.6% in northwestern Sud-Ubangi Province. Prevalence among boys (1.8%, 95% CI: 1.2%-2.7%) was double that among girls (0.7%, 95%CI: 0.4%-1.3%). Tetanus antibody-negativity, rurality, and lower household wealth were also significantly associated with higher HBsAg-positivity prevalence. We observed no difference in prevalence by age. Children had higher HBsAg-positivity odds if living with ≥1 HBsAg-positive adult household member (OR: 2.3, 95%CI: 0.7-7.8), particularly an HBsAg-positive mother (OR: 7.2, 95%CI:1.6-32.2)., Conclusion: In the largest national survey of HBV among children and household contacts in the DRC, we found that childhood HBV prevalence was 10-60 times the global target of 0.1%. We highlight specific regions and populations for further investigation and focused prevention efforts.

Background: The World Health Organization Africa region has high regional hepatitis B virus (HBV) prevalence, and evidence suggests more frequent horizontal HBV transmission than other regions. Context-specific epidemiological studies are needed to inform additional HBV prevention measures., Methods: In the cross-sectional Horizontal and Vertical Transmission of Hepatitis B (HOVER-HBV) study, we introduced HBV surface antigen (HBsAg) screening alongside existing HIV screening as part of routine antenatal care in high-volume maternity clinics in Kinshasa, Democratic Republic of Congo. We recruited households of pregnant women ("index mothers") who were HBsAg-positive and HBsAg-negative, defining households as index-positive and index-negative, respectively. Household members underwent HBsAg testing and an epidemiological survey. We evaluated HBsAg prevalence and potential transmission correlates., Results: We enrolled 1006 participants from 200 households (100 index-positive, 100 index-negative) across Kinshasa. HBsAg-positivity prevalence was more than twice as high in index-positive households (5.0% [95% confidence interval {CI}, 2.8%-7.1%]) as in index-negative households (1.9% [95% CI, .6%-3.2%]). HBsAg-positivity prevalence was 3.3 (95% CI, .9-11.8) times as high among direct offspring in index-positive versus index-negative households. Factors associated with HBsAg positivity included older age, marriage, and having multiple recent partners or any new sexual partners among index mothers; and older age, lower household wealth, sharing nail clippers, and using street salons among offspring in index-positive households., Conclusions: Vertical and horizontal HBV transmission within households is ongoing in Kinshasa. Factors associated with infection reveal opportunities for HBV prevention efforts, including perinatal prevention, protection during sexual contact, and sanitation of shared personal items., Competing Interests: Potential conflicts of interest. J. B. P. and P. T. report nonfinancial support from Abbott Laboratories (donation of hepatitis B laboratory testing and reagents for other studies), and J. B. P. reports consulting for Zymeron Corporation, all outside the submitted work. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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., Competing Interests: Conflict of interest All authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. IMPORTANCE Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens., Competing Interests: The authors declare no conflict of interest.

Background: Domesticated animal ownership is an understudied aspect of the human environment that influences mosquito biting behaviour and malaria transmission, and is a key part of national economies and livelihoods in malaria-endemic regions. In this study, we aimed to understand differences in Plasmodium falciparum prevalence by ownership status of common domesticated animals in DR Congo, where 12% of the world's malaria cases occur and anthropophilic Anopheles gambiae vectors predominate., Methods: In this cross-sectional study, we used survey data from individuals aged 15-59 years in the most recent (2013-14) DR Congo Demographic and Health Survey and previously performed Plasmodium quantitative real-time PCR (qPCR) to estimate P falciparum prevalence differences by household ownership of cattle; chickens; donkeys, horses, or mules; ducks; goats; sheep; and pigs. We used directed acyclic graphs to consider confounding by age, gender, wealth, modern housing, treated bednet use, agricultural land ownership, province, and rural location., Findings: Of 17 701 participants who had qPCR results and covariate data, 8917 (50·4%) of whom owned a domesticated animal, we observed large differences in malaria prevalence across types of animals owned in both crude and adjusted models. Household chicken ownership was associated with 3·9 (95% CI 0·6 to 7·1) more P falciparum infections per 100 people, whereas cattle ownership was associated with 9·6 (-15·8 to -3·5) fewer P falciparum infections per 100 people, even after accounting for bednet use, wealth, and housing structure., Interpretation: Our finding of a protective association conferred by cattle ownership suggests that zooprophylaxis interventions might have a role in DR Congo, possibly by drawing An gambiae feeding away from humans. Studies of animal husbandry practices and associated mosquito behaviours could reveal opportunities for new malaria interventions., Funding: The National Institutes of Health and the Bill & Melinda Gates Foundation., Translations: For the French and Lingala translations of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests JBP reports research support from Gilead Sciences, non-financial support from Abbott Laboratories, consulting for Zymeron Corporation, and honoraria from Virology Education, all outside the scope of this work. JBP also reports malaria research support from WHO, unrelated to this work. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, β-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens., Competing Interests: The authors declare no conflict of interest.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance to aminoglycoside-based drugs by actively extruding these noxious compounds out of E. coli cells. AcrD can also mediate resistance to a limited range of other amphiphilic agents, including bile acids, novobiocin, and fusidic acids. As there is no structural information available for any aminoglycoside-specific RND pump, here we describe cryo-electron microscopy (cryo-EM) structures of AcrD in the absence and presence of bound gentamicin. These structures provide new information about the RND superfamily of efflux pumps, specifically, that three negatively charged residues central to the aminoglycoside-binding site are located within the ceiling of the central cavity of the AcrD trimer. Thus, it is likely that AcrD is capable of picking up aminoglycosides via this central cavity. Through the combination of cryo-EM structural determination, mutagenesis analysis, and molecular simulation, we show that charged residues are critically important for this pump to shuttle drugs directly from the central cavity to the funnel of the AcrD trimer for extrusion. IMPORTANCE Here, we report cryo-EM structures of the AcrD aminoglycoside efflux pump in the absence and presence of bound gentamicin, posing the possibility that this pump is capable of capturing aminoglycosides from the central cavity of the AcrD trimer. The results indicate that AcrD utilizes charged residues to bind and export drugs, mediating resistance to these antibiotics.

The COVID-19 pandemic has disrupted access to, adherence to, and perceptions of routine vaccinations. We developed the Shift in Vaccine Confidence (SVC) survey tool to assess the impact of the pandemic on routine vaccinations, with a focus on the HBV vaccine, in Kinshasa, Democratic Republic of Congo (DRC). This study describes the content validation steps we conducted to ensure the survey tool is meaningful to measure changes in vaccine confidence to regular immunization (HBV vaccine) due to the pandemic. Three rounds of stakeholder feedback from a DRC-based study team, content and measurement experts, and study participants allowed us to produce a measure with improved readability and clarity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Alix Boisson reports a relationship with Drusilla L. Scott Summer Research Fellowship that includes: funding grants. Camille Johnson reports a relationship with National Institutes of Health that includes: funding grants. Angela Stover reports a relationship with Patient-Centered Outcomes Research Institute that includes: funding grants. Angela Stover reports a relationship with National Institutes of Health that includes: funding grants. Angela Stover reports a relationship with Agency for Healthcare Research and Quality that includes: funding grants. Angela Stover reports a relationship with Bladder Cancer Advocacy Network that includes: funding grants. Angela Stover reports a relationship with Sivan Innovations that includes: funding grants. Angela Stover reports a relationship with UroGen Pharma that includes: funding grants. Angela Stover reports a relationship with Association of Community Cancer Centers that includes: speaking and lecture fees. Angela Stover reports a relationship with Pfizer Inc that includes: speaking and lecture fees. Angela Stover reports a relationship with Genentech that includes: speaking and lecture fees. Angela Stover reports a relationship with Purchaser Business Group on Health that includes: funding grants. Angela Stover reports a relationship with Henry Ford Cancer Institute - Detroit that includes: speaking and lecture fees. Peyton Thompson reports a relationship with Novavax Inc that includes: funding grants. Peyton Thompson reports a relationship with National Institutes of Health that includes: funding grants. Peyton Thompson reports a relationship with American Society of Tropical Medicine and Hygiene that includes: funding grants. Jonathan B. Parr reports a relationship with Gilead Sciences Inc that includes: funding grants. Peyton Thompson reports a relationship with Gilead Sciences Inc that includes: funding grants. Jonathan B. Parr reports a relationship with Abbott Diagnostics that includes: non-financial support. Marcel Yotebieng reports a relationship with Abbott Diagnostics that includes: non-financial support. Peyton Thompson reports a relationship with Abbott Diagnostics that includes: non-financial support. Jonathan B. Parr reports a relationship with Virology Education that includes: speaking and lecture fees]., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The use of an integrated systems biology approach to investigate tissues and organs has been thought to be impracticable in the field of structural biology, where the techniques mainly focus on determining the structure of a particular biomacromolecule of interest. Here, we report the use of cryoelectron microscopy (cryo-EM) to define the composition of a raw bovine retinal pigment epithelium (RPE) lysate. From this sample, we simultaneously identify and solve cryo-EM structures of seven different RPE enzymes whose functions affect neurotransmitter recycling, iron metabolism, gluconeogenesis, glycolysis, axonal development, and energy homeostasis. Interestingly, dysfunction of these important proteins has been directly linked to several neurodegenerative disorders, including Huntington's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, and schizophrenia. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Optimal medical therapy for acute lower extremity deep venous thrombosis (DVT) remains an enigma. While clinical trials demonstrate non-inferiority with an oral anti-Xa inhibitor, or direct oral anticoagulant (DOAC), versus combined low-molecular weight heparin (LMWH) and oral vitamin K antagonist (VKA), the most effective regimen remains to be determined., Methods: This study is a single-center retrospective cohort study from October 2014 to December 2015 of patients with a diagnosis of acute DVT and subsequent serial lower extremity venous duplex. Demographics, medical history, medications, serial ultrasound findings, as well as the primary anticoagulant used for treatment were collected and analyzed by two independent data extractors. Treatment failure was defined as any new DVT or progression of an existing DVT within 3 months of diagnosis of the index clot. Risk factors for treatment failure were assessed using standard odds ratios and Fischer's exact test., Results: Among 496 patients with an acute lower extremity DVT, 54% ( n = 266) were men, mean age was 61 years, 35% ( n = 174) involved the popliteal or more proximal segments, and 442 had documentation of the primary treatment for DVT: 20% ( n = 90) received nothing; 20% ( n = 92) received an oral VKA; 34% ( n = 149) received a DOAC; 20% ( n = 90) received LMWH; and 5% ( n = 21) received another class of anticoagulant. Within 3 months, 21% ( n =89 out of 427) had treatment failure defined as any new DVT or progression of prior DVT. Patients treated with a DOAC were less likely to experience treatment failure when compared with any other treatment (odds ratio 0.43; 95% confidence intervals [0.23, 0.79]; p = 0.0069) and when compared with traditional oral VKA (OR 0.44; 95% CI [0.21, 0.92]; p = 0.029). None of prior history of DVT, pulmonary embolism, thrombophilia, renal insufficiency, hepatic insufficiency, cancer, or antiplatelet therapy correlated with treatment failure. Treatment outcome did not correlate with being on any anticoagulation versus none ( p = 0.74), nor did it correlate with the duration of treatment (<3 months versus ≥3 months) ( p = 0.42). Proximal and distal DVTs showed no difference in treatment failure (19% versus 22%, respectively; p = 0.43)., Conclusion: In summary, the use of a DOAC for acute lower extremity DVT yielded better overall outcomes and fewer treatment failures at 3 months as compared to traditional oral VKA therapy based on serial duplex imaging.

We recently developed a "Build and Retrieve" cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM maps for several individual proteins from a heterogeneous, multiprotein sample. Here we report the use of "Build and Retrieve" to define the composition of a raw human brain microsomal lysate. From this sample, we simultaneously identify and solve cryo-EM structures of five different brain enzymes whose functions affect neurotransmitter recycling, iron metabolism, glycolysis, axonal development, energy homeostasis, and retinoic acid biosynthesis. Interestingly, malfunction of these important proteins has been directly linked to several neurodegenerative disorders, such as Alzheimer's, Huntington's, and Parkinson's diseases. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level., (© 2022 Tringides et al.)

Background: Lower extremity amputations from diabetic foot ulcers (DFUs) are rebounding, and new biomarkers that predict wound healing are urgently needed. Anaerobic bacteria have been associated with persistent ulcers and may be a promising biomarker beyond currently recommended vascular assessments. It is unknown whether anaerobic markers are simply a downstream outcome of peripheral arterial disease (PAD) and ischemia, however. Here, we evaluate associations between two measures of anaerobic bacteria-abundance and metabolic activity-and PAD., Methods: We built a prospective cohort of 37 patients with baseline ankle brachial index (ABI) results. Anaerobic bacteria were measured in two ways: DNA-based total anaerobic abundance using 16S rRNA gene amplicon sequencing and resulting summed relative abundance, and RNA-based metabolic activity based on bacterial read annotation of metatranscriptomic sequencing. PAD was defined three ways: PAD diagnosis, ABI results, and a dichotomous definition of mild ischemia ( versus normal) based on ABI values. Statistical associations between anaerobes and PAD were evaluated using univariate odds ratios (ORs) or Spearman's correlations., Results: Total anaerobe abundance was not significantly associated with PAD diagnosis, ABI results, or mild ischemia (OR PAD  = 0.47, 95% CI = 0.023-7.23, p  = 0.60; Spearman's correlation coefficient ABI  = 0.24, p  = 0.17; OR mild ischemia  = 0.25, 95% CI = 0.005-5.86, p  = 0.42). Anaerobic metabolic activity was not significantly associated with PAD diagnosis, ABI results, or mild ischemia (OR PAD  = 1.99, 95% CI = 0.17-21.44, p  = 0.57; Spearman's correlation coefficient ABI  = 0.12, p  = 0.52; OR mild ischemia  = 0.90, 95% CI = 0.03-15.16, p  = 0.94)., Conclusion: Neither anaerobic abundance nor metabolic activity was strongly associated with our three definitions of PAD. Therefore, anaerobic bacteria may offer additional prognostic value when assessing wound healing potential and should be investigated as potential molecular biomarkers for DFU outcomes., Competing Interests: Competing interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2022.)

The interleukin-6 receptor antagonist tocilizumab became widely used early in the coronavirus disease 2019 (COVID-19) pandemic based on small observational studies that suggested clinical benefit in COVID-19 patients with a hyperinflammatory state. To inform our local treatment algorithms in the absence of randomized clinical trial results, we performed a rapid analysis of the first 11 hospitalized COVID-19 patients treated with tocilizumab at our academic medical center. We report their early clinical outcomes and describe the process by which we assembled a team of diverse trainees and stakeholders to extract, analyze, and disseminate data during a time of clinical uncertainty., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Hepatitis B virus (HBV) remains endemic throughout sub-Saharan Africa despite the widespread availability of effective childhood vaccines. In the Democratic Republic of the Congo, HBV treatment and birth-dose vaccination programmes are not established. We, therefore, aimed to evaluate the feasibility and acceptability of adding HBV testing and treatment of pregnant women as well as the birth-dose vaccination of HBV-exposed infants to the HIV prevention of mother-to-child transmission programme infrastructure in the Democratic Republic of the Congo., Methods: We did a feasibility study in two maternity centres in Kinshasa: Binza and Kingasani. Using the already established HIV prevention of mother-to-child transmission programme at these two maternity centres, we screened pregnant women for HBV infection at routine prenatal care registration. Those who tested positive and had a gestational age of 24 weeks or less were included in this study. Eligible pregnant women with a high viral load (≥200 000 IU/mL or HBeAg positivity, or both) were considered as having HBV of high risk of mother-to-child transmission and initiated on oral tenofovir disoproxil fumarate (300 mg/day) between 28 weeks and 32 weeks of gestation and continued through 12 weeks post partum. All HBV-exposed infants received a birth-dose of monovalent HBV vaccine (Euvax-B Pediatric: Sanofi Pasteur, Seoul, South Korea; 0·5 mL) within 24 h of life. All women were followed up for 24 weeks post partum, when they completed an exit questionnaire that assessed the acceptability of study procedures. The primary outcomes were the feasibility of screening pregnant women to identify those at high risk for HBV mother-to-child transmission and to provide them with antiviral prophylaxis, the feasibility of administrating the birth-dose vaccine to exposed infants, and the acceptability of this prevention programme. This study is registered with ClinicalTrials.gov, NCT03567382., Findings: Between Sept 24, 2018, and Feb 22, 2019, 4016 women were approached and screened. Of these pregnant women, 109 (2·7%) were positive for HBsAg. Of the 109 women, 91 (83%) met the eligibility criteria for participation. However, only data from 90 women-excluding one woman who had a false pregnancy-were included in the study analysis. The median overall age of the enrolled women was 31 years (IQR 25-34) and the median overall gestational age was 19 weeks (15-22). Ten (11%) of 91 women evaluated had high-risk HBV infection. Nine (90%) of the ten pregnant women with high-risk HBV infection received tenofovir disoproxil fumarate and one (10%) refused therapy and withdrew from the study; five (56%) of the nine women achieved viral suppression (ie, <200 000 IU/mL) on tenofovir disoproxil fumarate therapy by the time of delivery and the remaining four (44%) had decreased viral loads from enrolment to delivery. A total of 88 infants were born to the 90 enrolled women. Of the 88 infants, 60 (68%) received a birth-dose vaccine; of these, 46 (77%) received a timely birth-dose vaccine. No cases of HBV mother-to-child transmission were observed. No serious adverse events associated with tenofovir disoproxil fumarate nor with the birth-dose vaccine were reported. Only one (11%) of nine women reported dizziness during the course of tenofovir disoproxil fumarate therapy. The study procedures were considered highly acceptable (>80%) among mothers., Interpretation: Adding HBV screening and treatment of pregnant women and infant birth-dose vaccination to existing HIV prevention of mother-to-child transmission platforms is feasible in countries such as the Democratic Republic of the Congo. Birth-dose vaccination against HBV infection integrated within the current Expanded Programme on Immunisation and HIV prevention of mother-to-child transmission programme could accelerate progress toward HBV elimination in Africa., Funding: Gillings Innovation Laboratory award and the National Institutes of Health., Translations: For the French and Lingala translations of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests PT and JBP report support from the American Society of Tropical Medicine and Hygiene–Burroughs Wellcome Fund awards, outside the submitted work. PT, RJ, and JBP report research support from Gilead Sciences, outside the submitted work. JBP reports grants from the US National Institutes of Health (NIH), outside the submitted work. CEM reports a grant from the Infectious Diseases Society of America, outside the submitted work. RJ reports consulting fees from Dynavax, outside the submitted work; membership on the American Association for the Study of Liver Diseases (AASLD)–Infections Diseases Society of America Hepatitis C Virus Guidelines panel and the AASLD Viral Hepatitis Elimination Task Force; and a stipend from Elsevier for editorial services as Co-Editor-in-Chief of Clinical Therapeutics. GC is an employee and shareholder of Abbott Laboratories. JBP reports research support from WHO and honoraria from Virology Education, outside the submitted work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall., Competing Interests: The authors have declared that no competing interests exist.

Contaminated drinking water causes morbidity and mortality worldwide, especially in low- and middle-income countries. Drinking water quality has been studied extensively in household settings, but little research is available on drinking water quality in schools. School settings are of particular importance, because children are more susceptible than adults to a variety of diseases from contaminated drinking water. Many school water, sanitation and hygiene (WaSH) interventions have been studied for their efficacy to reduce diarrheal disease incidence, but few have evaluated drinking water quality, which reflects an important exposure pathway between WaSH services and health outcomes. Using school surveys developed from internationally established WaSH indicators and field microbiological water quality tests, we studied 374 rural schools in Mozambique and Uganda to understand the association between specific WaSH services and drinking water microbiological contamination, specifically testing most probable number (MPN) of Escherichia coli, an indicator of fecal contamination, per 100 mL. In Mozambique and Uganda, 71% and 83% respectively of rural schools had low risk drinking water quality (<1 E. coli/100 mL); thirteen percent and seven percent had very high-risk water quality (≥100 E. coli/100 mL). When accounting for all WaSH services studied, schools that used an improved-type water source had 0.22 times less E. coli in stored drinking water in Mozambique (95% CI: 0.07, 0.65) and 0.12 times less E. coli in Uganda (95% CI: 0.02, 0.80). In Mozambique, use of a water source within 30 minutes for travel and collection and the presence of water and soap/ash for handwashing were also significantly associated with less E. coli in drinking water. The findings of this study provide public health practitioners with implementable WaSH services to improve school drinking water quality, which has implications for the health, learning environment, and cognitive development of school children in rural Mozambique and Uganda., (Copyright © 2021. Published by Elsevier GmbH.)

Antibiotic-resistant strains of the Gram-negative pathogen Acinetobacter baumannii have emerged as a significant global health threat. One successful therapeutic option to treat bacterial infections has been to target the bacterial ribosome. However, in many cases, multidrug efflux pumps within the bacterium recognize and extrude these clinically important antibiotics designed to inhibit the protein synthesis function of the bacterial ribosome. Thus, multidrug efflux within A. baumannii and other highly drug-resistant strains is a major cause of failure of drug-based treatments of infectious diseases. We here report the first structures of the A cinetobacter d rug e fflux (Ade)J pump in the presence of the antibiotic eravacycline, using single-particle cryo-electron microscopy (cryo-EM). We also describe cryo-EM structures of the eravacycline-bound forms of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. Our data indicate that the AdeJ pump primarily uses hydrophobic interactions to bind eravacycline, while the 70S ribosome utilizes electrostatic interactions to bind this drug. Our work here highlights how an antibiotic can bind multiple bacterial targets through different mechanisms and potentially enables drug optimization by taking advantage of these different modes of ligand binding. IMPORTANCE Acinetobacter baumannii has developed into a highly antibiotic-resistant Gram-negative pathogen. The prevalent AdeJ multidrug efflux pump mediates resistance to different classes of antibiotics known to inhibit the function of the 70S ribosome. Here, we report the first structures of the A. baumannii AdeJ pump, both in the absence and presence of eravacycline. We also describe structures of the A. baumannii ribosome bound by this antibiotic. Our results indicate that AdeJ and the ribosome use very distinct binding modes for drug recognition. Our work will ultimately enable structure-based drug discovery to combat antibiotic-resistant A. baumannii infection.

Multidrug resistant (MDR) bacteria are a global threat with many common infections becoming increasingly difficult to eliminate. While significant effort has gone into the development of potent biocides, the effectiveness of many first-line antibiotics has been diminished due to adaptive resistance mechanisms. Bacterial membrane proteins belonging to the resistance-nodulation-cell division (RND) superfamily play significant roles in mediating bacterial resistance to antimicrobials. They participate in multidrug efflux and cell wall biogenesis to transform bacterial pathogens into "superbugs" that are resistant even to last resort antibiotics. In this review, we summarize the RND superfamily of efflux transporters with a primary focus on the assembly and function of the inner membrane pumps. These pumps are critical for extrusion of antibiotics from the cell as well as the transport of lipid moieties to the outer membrane to establish membrane rigidity and stability. We analyze recently solved structures of bacterial inner membrane efflux pumps as to how they bind and transport their substrates. Our cumulative data indicate that these RND membrane proteins are able to utilize different oligomerization states to achieve particular activities, including forming MDR pumps and cell wall remodeling machineries, to ensure bacterial survival. This mechanistic insight, combined with simulated docking techniques, allows for the design and optimization of new efflux pump inhibitors to more effectively treat infections that today are difficult or impossible to cure.

The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen's kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

Antibiotic resistance is an emerging threat to global health. Current treatment regimens for these types of bacterial infections are becoming increasingly inadequate. Thus, new innovative technologies are needed to help identify and characterize novel drugs and drug targets which are critical in order to combat multidrug-resistant bacterial strains. Bacterial efflux systems have emerged as an attractive target for drug design, as blocking their export function significantly increases the potency of administered antibiotics. However, in order to develop potent and tolerable efflux pump inhibitors with high efficacy, detailed structural information is required for both the apo- and substrate-bound forms of these membrane proteins. The emergence of cryo-electron microscopy (cryo-EM) has greatly advanced the field of membrane protein structural biology. It has significantly enhanced the ability to solve large multi-protein complexes as well as extract meaningful data from a heterogeneous sample, such as identification of several assembly states of the bacterial ribosome, from a single data set. This technique can be expanded to solve the structures of substrate-bound efflux pumps and entire efflux systems from previously unusable membrane protein sample preparations. Subsequently, cryo-EM combined with other biophysical techniques has the potential to markedly advance the field of membrane protein structural biology. The ability to discern complete transport machineries, enzymatic signal transduction pathways, and other membrane-associated complexes will help us fully understand the complexities of the membrane proteome., Competing Interests: The authors declare that they have no competing interests.Competing interests: No competing interests were disclosed.Competing interests: No competing interests were disclosed.Competing interests: No competing interests were disclosed.Competing interests: No competing interests were disclosed.Competing interests: No competing interests were disclosed., (Copyright: © 2021 Yu EW et al.)

Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent A cinetobacterd rug e fflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into "dimer plus monomer" and "monomer plus monomer plus monomer" configurations. We also discover that a single AdeB protomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dynamics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently. IMPORTANCE Acinetobacter baumannii has emerged as one of the most highly antibiotic-resistant Gram-negative pathogens. The prevalent AdeB multidrug efflux pump mediates resistance to a broad spectrum of clinically relevant antimicrobial agents. Here, we report six cryo-EM structures of the trimeric AdeB pump in the presence of ethidium bromide. We discover that a single AdeB protomer can simultaneously anchor a number of ligands, and different AdeB protomers can bind ethidium molecules simultaneously. The results indicate that each AdeB protomer within the trimer recognizes and extrudes drugs independently., (Copyright © 2021 Morgan et al.)

Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a number of resistance determinants to evade current antibiotics. One of the major resistance mechanisms employed by these pathogens is the use of multidrug efflux pumps. These pumps extrude xenobiotics directly out of bacterial cells, resulting in treatment failures when common antibiotics are administered. Here, the structure of the novel tetracycline antibiotic TP-6076, bound to both the A cinetobacter d rug e fflux pump AdeJ and the ribosome from Acinetobacter baumannii, using single-particle cryo-electron microscopy (cryo-EM), is elucidated. In this work, the structure of the AdeJ-TP-6076 complex is solved, and we show that AdeJ utilizes a network of hydrophobic interactions to recognize this fluorocycline. Concomitant with this, we elucidate three structures of TP-6076 bound to the A. baumannii ribosome and determine that its binding is stabilized largely by electrostatic interactions. We then compare the differences in binding modes between TP-6076 and the related tetracycline antibiotic eravacycline in both targets. These differences suggest that modifications to the tetracycline core may be able to alter AdeJ binding while maintaining interactions with the ribosome. Together, this work highlights how different mechanisms are used to stabilize the binding of tetracycline-based compounds to unique bacterial targets and provides guidance for the future clinical development of tetracycline antibiotics. IMPORTANCE Treatment of antibiotic-resistant organisms such as A. baumannii represents an ongoing issue for modern medicine. The multidrug efflux pump AdeJ serves as a major resistance determinant in A. baumannii through its action of extruding antibiotics from the cell. In this work, we use cryo-EM to show how AdeJ recognizes the experimental tetracycline antibiotic TP-6076 and prevents this drug from interacting with the A. baumannii ribosome. Since AdeJ and the ribosome use different binding modes to stabilize interactions with TP-6076, exploiting these differences may guide future drug development for combating antibiotic-resistant A. baumannii and potentially other strains of MDR bacteria.

Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.

Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii , a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections. IMPORTANCE Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen., (Copyright © 2020 Morgan et al.)

Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle. IMPORTANCE Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Background: Current procedures to treat severe atherosclerosis are traumatic to the arterial wall and often result in restenosis due to neointimal hyperplasia. We developed a novel therapy using a specially designed double occlusion balloon catheter, ultrasonic wire, and enzymatic digestion solution to atraumatically debulk atherosclerotic plaques., Materials and Methods: A combination of different enzymes, chemicals, and treatment conditions were evaluated for its effect at reducing atherosclerotic plaque harvested from human carotid artery endarterectomies ex vivo. The optimized digestion solution was examined in harvested intact human superficial femoral arteries in situ. A conventional Yorkshire/Landrace and a genetically modified Yucatan minipig homozygous for a nonfunctional LDLR mutation were used to evaluate the endovascular therapy in nonatherosclerotic and atherosclerotic environments in vivo., Results: Ex vivo, the technology successfully digested human carotid artery plaques by 75%. In situ, the therapy successfully reduced plaque area in harvested superficial femoral arteries by 46%. In vivo, the endovascular therapy was technically feasible and demonstrated initial safety with no thrombosis, dissection, or aneurysmal dilatation in a nonatherosclerotic porcine model. In an atherosclerotic porcine model, the therapy demonstrated initial efficacy by successfully reducing atherosclerotic plaque while preserving the arterial wall with an intact internal elastic lamina., Conclusions: Using human plaque, human artery, and a normal and atherosclerotic pig model, we demonstrated that delivery of our therapy to the vasculature is technically feasible, appears safe, and shows initial efficacy. Our percutaneous plaque debulking method is a unique and promising therapy for the treatment of atherosclerosis and warrants further study., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: Isolated spontaneous dissection of the superior mesenteric artery (SMA) and celiac artery (CA) remains a rare condition; however, it has been increasingly noted incidentally on diagnostic imaging. The purpose of this study was to examine the natural history and outcomes of patients presenting with isolated spontaneous mesenteric artery dissection (SMAD). We hypothesized that most SMADs can be treated nonoperatively., Methods: This was a single-center retrospective review of patients presenting with the diagnosis of SMAD between 2006 and 2016. Data analysis included demographics, clinical data, radiologic review, treatment, and outcomes., Results: A total of 77 patients were found to have CA dissection, SMA dissection, or both in the absence of aortic dissection diagnosed on computed tomography or magnetic resonance imaging. The average age was 56 years (range, 26-86 years), 80% were male, and 10 patients (13%) had underlying connective tissue disorders. The majority, 64%, presented with symptoms including abdominal pain, back pain, and chest pain; the remaining 36% were asymptomatic. Combined SMA and CA dissection was found in 14 (18%) patients; 33 (43%) presented with isolated CA dissection, and 30 (39%) presented with isolated SMA dissection. Only four patients required intervention. Mesenteric bypass was performed in two patients, and SMA endarterectomy with patch angioplasty was performed in one patient for signs of bowel ischemia. No patient required bowel resection. The two bypasses were anastomosed to a branch of the SMA, and complete lumen restoration was seen on long-term imaging follow-up. One patient underwent stent grafting of the CA and hepatic artery for aneurysmal degeneration 1 month after diagnosis. The remaining 73 patients were managed nonoperatively; 40 (52%) were treated with a short course of anticoagulation, 23 (30%) were treated with antiplatelet therapy, and 10 (13%) were treated with observation alone. No other late interventions or recurrences were noted during a mean follow-up of 21 months., Conclusions: Whereas isolated SMAD poses a risk of visceral ischemia, most patients presenting with this diagnosis can be treated nonoperatively with a short course of antiplatelet or anticoagulant therapy. Only a small number of patients require surgical revascularization for bowel ischemia., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Enteroviruses use a type I Internal Ribosome Entry Site (IRES) structure to facilitate protein synthesis and promote genome replication. Type I IRES elements require auxiliary host proteins to organize RNA structure for 40S ribosomal subunit assembly. Heterogeneous nuclear ribonucleoprotein A1 stimulates enterovirus 71 (EV71) translation in part through specific interactions with its stem loop II (SLII) IRES domain. Here, we determined a conjoined NMR-small angle x-ray scattering structure of the EV71 SLII domain and a mutant that significantly attenuates viral replication by abrogating hnRNP A1 interactions. Native SLII adopts a locally compact structure wherein stacking interactions in a conserved 5'-AUAGC-3' bulge preorganize the adjacent helices at nearly orthogonal orientations. Mutating the bulge sequence to 5'-ACCCC-3' ablates base stacking in the loop and globally reorients the SLII structure. Biophysical titrations reveal that the 5'-AUAGC-3' bulge undergoes a conformational change to assemble a functional hnRNP A1-RNA complex. Importantly, IRES mutations that delete the bulge impair viral translation and completely inhibit replication. Thus, this work provides key details into how an EV71 IRES structure adapts to hijack a cellular protein, and it suggests that the SLII domain is a potential target for antiviral therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants ( n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5'-YAG-3' and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

Objective: To describe the components of targeted nanotherapeutics and to review their applications in the treatment of surgical diseases., Background: Targeted nanotherapeutic is a novel strategy for treating a variety of diseases and is an emerging technology that offers advantages over current treatment strategies. The nanoscale size, combined with the ability to surface functionalize the delivery vehicle to enable targeting and incorporate a therapeutic payload, provides a new and innovative therapeutic platform to treat surgical diseases that has yet to be fully realized in the surgical arena., Methods: A comprehensive literature review of nanotherapeutics, targeting strategies, and their utility in treating surgical diseases is performed., Results: Targeted nanotherapeutics have demonstrated safety and biocompatibility in treating surgical diseases. The ability to surface functionalize the nanoparticles affords a unique tailorability that enables targeting specificity and therapeutic payload delivery to treat a variety of surgical diseases. Moreover, the small size and targeting capabilities allow access to biological compartments, such as the blood-brain barrier, that have previously been difficult to treat., Conclusions: Targeted nanotherapeutics represent a novel therapeutic platform and have great potential to impact the treatment of surgical diseases.

Aims: Cardiovascular interventions continue to fail as a result of arterial restenosis secondary to neointimal hyperplasia. We sought to develop and evaluate a systemically delivered nanostructure targeted to the site of arterial injury to prevent neointimal hyperplasia. Nanostructures were based on self-assembling biodegradable molecules known as peptide amphiphiles. The targeting motif was a collagen-binding peptide, and the therapeutic moiety was added by S-nitrosylation of cysteine residues., Results: Structure of the nanofibers was characterized by transmission electron microscopy and small-angle X-ray scattering. S-nitrosylation was confirmed by mass spectrometry, and nitric oxide (NO) release was assessed electrochemically and by chemiluminescent detection. The balloon carotid artery injury model was performed on 10-week-old male Sprague-Dawley rats. Immediately after injury, nanofibers were administered systemically via tail vein injection. S-nitrosylated (S-nitrosyl [SNO])-targeted nanofibers significantly reduced neointimal hyperplasia 2 weeks and 7 months following balloon angioplasty, with no change in inflammation., Innovation: This is the first time that an S-nitrosothiol (RSNO)-based therapeutic was shown to have targeted local effects after systemic administration. This approach, combining supramolecular nanostructures with a therapeutic NO-based payload and a targeting moiety, overcomes the limitations of delivering NO to a site of interest, avoiding undesirable systemic side effects., Conclusion: We successfully synthesized and characterized an RSNO-based therapy that when administered systemically, targets directly to the site of vascular injury. By integrating therapeutic and targeting chemistries, these targeted SNO nanofibers provided durable inhibition of neointimal hyperplasia in vivo and show great potential as a platform to treat cardiovascular diseases.

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3' acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)