Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli.

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.

Live-Attenuated Vaccines in Pediatric Solid Organ Transplant.

Measles, mumps, rubella (MMR), and varicella incidence rates have increased due to the delayed vaccination schedules of children secondary to the COVID-19 pandemic. Decreased herd immunity creates a risk for immunocompetent children and immunocompromised individuals in the community. Historically, live-attenuated vaccines (MMR and varicella) were recommended before solid organ transplants. The amount of time before transplant when this is appropriate is often debated, as is the utility of vaccine titers. MMR and varicella vaccines previously were not recommended in immunocompromised patients post-solid organ transplant due to the undue risk of transmission and posed infection risk. The new literature on live-attenuated vaccines in post-transplant pediatric patients provides more insight into the vaccines' safety and efficacy. The present article aims to provide guidance on live-attenuated vaccines (MMR and varicella) in the pre-transplant and post-operative solid organ transplant phases of care in pediatric patients.

The acute pain crisis in sickle cell disease: What can be done to improve outcomes?

The acute pain crisis (APC) is the commonest complication of sickle cell disease (SCD). Severe episodes may require treatment in hospital with strong opioid analgesic drugs, combined with additional supportive care measures. Guidelines for APC management have been produced over the past two decades gathering evidence from published studies, expert opinion, and patient perspective. Unfortunately, reports from multiple sources indicate that guidelines are often not followed, and that acute care in emergency departments and on acute medical wards is suboptimal. It is important to understand what leads to this breakdown in health care, and to identify evidence-based interventions which could be implemented to improve care. This review focuses on recently published articles as well as information about on-going clinical trials. Aspects of care which could potentially make a difference to patient experience include availability and accessibility of individual care plans agreed between patient and treating specialist, innovative means of delivering initial opioids to reduce time to first analgesia, and availability of a specialist unit away from the ED, where expert care can be delivered in a more compassionate environment. The current evidence of improved outcomes and health economic advantage with these interventions is inadequate, and this is hampering their implementation into health care systems.

Coumarins-lipophilic cations conjugates: Efficient mitocans targeting carbonic anhydrases.

Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative agents. We have accomplished the conjugation of a coumarin fragment with lipophilic cations (triphenylphosphonium salts, guanidinium) for providing mitochondriotropic agents that simultaneously target also carbonic anhydrases IX and XII, involved in the development and progression of cancer. The new compounds prepared herein turned out to be strong inhibitors of carbonic anhydrases IX and XII of human origin (low-to-mid nM range), also endowed with high selectivity, exhibiting negligible activity towards cytosolic CA isoforms. Key interactions with the enzyme were analysed using docking and molecular dynamics simulations. Regarding their in vitro antiproliferative activities, an increase of the tether length connecting both pharmacophores led to a clear improvement in potency, reaching the submicromolar range for the lead compounds, and an outstanding selectivity towards tumour cell lines (S.I. up to >357). Cytotoxic effects were also analysed on MDR cell lines under hypoxic and normoxic conditions. Chemoresistance exhibited by phosphonium salts, and not by guanidines, against MDR cells was based on the fact that the former were found to be substrates of P-glycoprotein (P-gp), the pump responsible for extruding foreign chemicals; this situation was reversed by administrating tariquidar, a third generation P-gp inhibitor. Moreover, phosphonium salts provoked a profound depolarization of mitochondria membranes from tumour cells, thus probably compromising their oxidative metabolism. To gain insight into the mode of action of title compounds, continuous live cell microscopy was employed; interestingly, this technique revealed two different antiproliferative mechanisms for both families of mitocans. Whereas phosphonium salts had a cytostatic effect, blocking cell division, guanidines led to cell death via apoptosis.

A Fast, Reproducible, High-throughput Variant Calling Workflow for Population Genomics.

The increasing availability of genomic resequencing data sets and high-quality reference genomes across the tree of life present exciting opportunities for comparative population genomic studies. However, substantial challenges prevent the simple reuse of data across different studies and species, arising from variability in variant calling pipelines, data quality, and the need for computationally intensive reanalysis. Here, we present snpArcher, a flexible and highly efficient workflow designed for the analysis of genomic resequencing data in nonmodel organisms. snpArcher provides a standardized variant calling pipeline and includes modules for variant quality control, data visualization, variant filtering, and other downstream analyses. Implemented in Snakemake, snpArcher is user-friendly, reproducible, and designed to be compatible with high-performance computing clusters and cloud environments. To demonstrate the flexibility of this pipeline, we applied snpArcher to 26 public resequencing data sets from nonmammalian vertebrates. These variant data sets are hosted publicly to enable future comparative population genomic analyses. With its extensibility and the availability of public data sets, snpArcher will contribute to a broader understanding of genetic variation across species by facilitating the rapid use and reuse of large genomic data sets.

Organ agar serves as physiologically relevant alternative for in vivo bacterial colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to bacterial colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. Organ agar was also useful for identifying previously unknown links between biosynthetic genes and swarming motility. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

Conserved metabolic regulator ArcA responds to oxygen availability, iron limitation, and cell envelope perturbations during bacteremia.

IMPORTANCE: Infections of the bloodstream are life-threatening and can result in sepsis. Gram-negative bacteria cause a significant portion of bloodstream infections, which is also referred to as bacteremia. The long-term goal of our work is to understand how such bacteria establish and maintain infection during bacteremia. We have previously identified the transcription factor ArcA, which promotes fermentation in bacteria, as a likely contributor to the growth and survival of bacteria in this environment. Here, we study ArcA in the Gram-negative species Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens. Our findings aid in determining how these bacteria sense their environment, utilize nutrients, and generate energy while countering the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

Males with sickle cell disease have higher risks of cerebrovascular disease, increased inflammation, and a reduced response to hydroxyurea.

Biological sex is important. Male sex is associated with worse outcomes in most measures, including cerebrovascular disease, hospital admissions, and blood transfusions, but not survival. Females also appear to have a better response to hydroxyurea therapy, reduced markers of inflammation, and better liver function.

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress.

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD supported liver fitness in vivo and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.

Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors.

The involvement of carbonic anhydrases (CAs) in a myriad of biological events makes the development of new inhibitors of these metalloenzymes a hot topic in current Medicinal Chemistry. In particular, CA IX and XII are membrane-bound enzymes, responsible for tumour survival and chemoresistance. Herein, a bicyclic carbohydrate-based hydrophilic tail (imidazolidine-2-thione) has been appended to a CA-targeting pharmacophore (arylsulfonamide, coumarin) with the aim of studying the influence of the conformational restriction of the tail on the CA inhibition. For this purpose, the coupling of sulfonamido- or coumarin-based isothiocyanates with reducing 2-aminosugars, followed by the sequential acid-promoted intramolecular cyclization of the corresponding thiourea and dehydration reactions, afforded the corresponding bicyclic imidazoline-2-thiones in good overall yield. The effects of the carbohydrate configuration, the position of the sulfonamido motif on the aryl fragment, and the tether length and substitution pattern on the coumarin were analysed in the in vitro inhibition of human CAs. Regarding sulfonamido-based inhibitors, the best template turned out to be a d-galacto-configured carbohydrate residue, meta-substitution on the aryl moiety (9b), with Ki against CA XII within the low nM range (5.1 nM), and remarkable selectivity indexes (1531 for CA I and 181.9 for CA II); this provided an enhanced profile in terms of potency and selectivity compared to more flexible linear thioureas 1-4 and the drug acetazolamide (AAZ), used herein as a reference compound. For coumarins, the strongest activities were found for substituents devoid of steric hindrance (Me, Cl), and short linkages; derivatives 24h and 24a were found to be the most potent inhibitors against CA IX and XII, respectively (Ki = 6.8, 10.1 nM), and also endowed with outstanding selectivity (Ki > 100 µM against CA I, II, as off-target enzymes). Docking simulations were conducted on 9b and 24h to gain more insight into the key inhibitor-enzyme interactions.

Organ agar serves as physiologically relevant alternative for in vivo colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

A Multi-Topic Bystander Intervention Program for Upper-Level Undergraduate Students: Outcomes in Sexual Violence, Racism, and High-Risk Alcohol Situations.

Few bystander intervention trainings programs have evaluated behavioral outcomes in previously trained upper-level undergraduate students. Rigorous study designs are necessary to understand how multi-topic programs influence student outcomes to intervene against sexual violence, racism, and high-risk alcohol situations. A single-session bystander training for emphasizing communication strategies was developed for juniors and seniors on a private, Midwestern college campus. The training addressed sexual violence, racism, and high-risk alcohol situations and was evaluated using a randomized waitlist-control design in student-housing units. Online Qualtrics surveys were completed by 101 student participants (57 in the intervention group and 44 in the control group). Students responded to nine harm scenarios involving sexual violence, racism, and high-risk alcohol situations at baseline and 7 weeks follow-up. Between-group changes in scores were compared to determine the effect of the program on students' (a) readiness to intervene; (b) confidence to intervene; (c) bystander behaviors among students who witnessed actual or potentially harmful events; and (d) bystander reports of their experiences. Qualitative analysis assessed how the program influenced the use of positive verbal communication strategies. Program effects increased positive bystander experiences when helping someone who had too much to drink and needed assistance. Over time both groups reported increased confidence levels to intervene when someone intoxicated was being isolated with sexual intent. There were no further significant findings in readiness, confidence, behaviors, or other experiences, though some positive nonsignificant trends emerged. The program demonstrated little efficacy. Results suggest opportunities to improve bystander outcomes in low-risk primary prevention situations and racist scenarios, suggesting that targeted intervention of these outcomes may be useful when developing programming with previously-trained students. As universities expand prevention work beyond the first year, lessons learned may help inform multi-year programming across health topics to prevent harm and create healthier college campuses.

Speech-language disorder severity, academic success, and socioemotional functioning among multilingual and English children in the United States: The National Survey of Children's Health.

Research points to negative associations between educational success, socioemotional functioning, and the severity of symptoms in some speech-language disorders (SLDs). Nonetheless, the majority of studies examining SLDs in children have focused on monolinguals. More research is needed to determine whether the scant findings among multilinguals are robust. The present study used parent report data from the U.S. National Survey of Children's Health (2018 to 2020) to gain a better understanding of the impacts of SLD severity on indicators of academic success and socioemotional functioning among multilingual (n = 255) and English monolingual (n = 5,952) children with SLDs. Tests of between-group differences indicated that multilingual children evidenced more severe SLDs, had lower school engagement, and had lower reports of flourishing than English monolingual children with SLDs. Further, a greater proportion of multilingual children with SLDs missed more school days than English monolinguals. However, multilinguals were less likely to bully others or have been bullied than monolinguals. While the previous between-group differences were statistically significant, they were small (vs ≤ 0.08). Increased SLD severity predicted an increased number of repeated school grades, increased absenteeism, and decreased school engagement, when age and socioeconomic status were controlled. Increased SLD severity also predicted greater difficulty making and keeping friends and decreased flourishing. The effect of SLD severity on being bullied was statistically significant for the monolinguals but not multilinguals. There was a statistically significant interaction for SLD severity and sex for school engagement and difficulty making and keeping friends for monolinguals but not multilinguals. The interactions indicated that school engagement decreased more for females than for males while difficulties making and keeping friends increased more for males than females as one's SLD severity increased. While some findings were specific to monolinguals, tests of measurement invariance indicated that the same general pattern of relations among the variables were evident across the groups of multilinguals and monolinguals. These final findings can inform the interpretation of the results from both the current and future studies, while the overall findings can inform the development of intervention programs, thereby improving the long-term academic and socioemotional outcomes of children with SLDs.

Comparative study highlights how gene flow shapes adaptive genomic architecture.

Adaptation to environmental conditions, and the mechanisms underlying these adaptations, can vary greatly among species. This variation can be attributed to a variety of factors including the strength of evolutionary processes like selection, gene flow, time since divergence, and/or genetic drift, as well as the interactions between these processes. A number of simulation and theoretical studies have helped elucidate the role of these processes on the genomic basis of adaptation (Schaal et al., 2022; Yeaman et al., 2016). However, complementary empirical studies to test these theoretical expectations for within-species adaptation have been limited due to the challenging nature of evaluating these processes in a comparative framework. To do this effectively, it is necessary to have systems where the range of environmental variation is similar between species, but where one or more of these evolutionary processes vary. In a From the Cover article in this issue of Molecular Ecology, Shi et al. (2022) provide an excellent example of a freshwater system where rates of gene flow differ between populations of six riverine species due to variation in spawning strategies (i.e., broadcast spawners = high gene flow, nest spawners = low gene flow), but all experience the same variation in environmental conditions across their distributions. The authors take a multivariate approach to evaluate the genomic basis of adaptation by using a combination of differentiation-based and genotype-environment association (GEA) methods. By comparing the amount of gene flow between species and the resulting genomic basis of local adaptation, they are able to infer how genomic architecture may be shaped by rates of gene flow. Their results identify a general pattern of increased genomic clustering in species with increasing levels of gene flow. However, two of six species did not follow this pattern, which could be due to additional factors not assessed. Additionally, they provide convincing evidence that the underlying evolutionary mechanisms that formed genomic clusters within each species vary. These deviations from a general pattern highlight how difficult evaluating these processes in natural populations are, particularly because species-specific responses can vary dramatically. Taken together, their comparative framework for assessing the genomic architecture of adaptation is unique, sheds important light on how evolutionary processes can impact adaptation, and provides robust empirical support of foundational theoretical and simulation studies.

Synthesis, antiproliferative evaluation and in silico studies of a novel steroidal spiro morpholinone.

Estrogens play a pivotal role in the development of estrogen-dependent breast cancer and other hormone-dependent disorders. A common strategy to overcome the pathological effects of estrogens is the use of aromatase inhibitors (AIs), which bind to the enzyme and prevent the union with the natural substrate, decreasing the amount of estrogens produced. Several AIs have been developed, including inhibitors with a steroidal backbone and a nitrogen heterocycle in their structure. Encouraged by the notable results presented by current and clinical steroidal drugs, herein we present the synthesis of a steroidal spiro morpholinone derivative as a plausible aromatase inhibitor. The morpholinone derivative was synthesized over a six-step methodology starting from estrone. The title compound and its hydroxychloroacetamide derivative precursor were evaluated for their antiproliferative profile against estrogen-dependent and independent solid tumor cell lines: A549, HBL-100, HeLa, SW1573, T-47D and WiDr. Both compounds exhibited a potent antiproliferative activity in the micromolar range against the six cancer cell lines, with the hydroxychloroacetamide derivative precursor being a more potent inhibitor (GI50 = 0.25-2.4 µM) than the morpholinone derivative (GI50 = 2.0-11 µM). Furthermore, both compounds showed, in almost all cases, better GI50 values than the steroidal anticancer drugs abiraterone and galeterone. Docking simulations of the derivatives were performed in order to explain the experimental biological activity. The results showed interactions with the iron heme (derivative 3) and important residues of the steroidal binding-site (Met374) for the inhibition of human aromatase. A correlation was found between in vitro assays and the score obtained from the molecular docking study.

The Klebsiella pneumoniae ter Operon Enhances Stress Tolerance.

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term-care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to radical oxygen species-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify the genes that are required to resist K2TeO3-induced and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3 disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as a stress tolerance factor, thereby explaining its role in enhancing fitness in the gut and bladder.

An Overview of Solid Organ Transplantation in Patients With Sickle Cell Disease.

Sickle cell disease is a common genetic disorder affecting >300 000 people across the world. The vast majority of patients cared for in high-resource settings live well into adulthood, but many develop a high burden of disease complications. Good standard of care including disease-modifying agents and transfusion programs limits the number of patients who develop end-stage organ disease, but for those that do, the prognosis can be very poor. Solid organ transplantation is a well-established mode of treatment for patients with sickle cell disease and kidney or liver failure, but appropriate patient selection and perioperative management are important for achieving good outcomes. Hematopoietic stem cell transplantation and gene therapy may offer novel treatment options for adult patients with chronic organ damage in the future, but these are not yet widely available. For now, good, holistic care and early intervention of end-organ complications can minimize the number of patients requiring solid organ transplantation later in life.

Overall Survival in Patients with Stage IV Pan-NET Eligible for Liver Transplantation.

BACKGROUND: The use of liver transplantation (LT) in patients with stage IV neuroendocrine pancreatic tumors (pan-NET) is under debate. Previous studies report a 5-year survival of 27-53% after LT in pan-NET and up to 92.7% in patients with mixed NETs. This study aimed to determine survival rates of patients with stage IV pan-NET meeting criteria for LT while only subjected to multimodal treatment. METHODS: Medical records of patients with pan-NET diagnosed from 2000 to 2021 at a tertiary referral center were evaluated for eligibility. Patients without liver metastases, who did not undergo primary tumor surgery, age > 75 years and with grade 3 tumors were excluded. The patients were divided into groups; all included patients, patients meeting the Milan, the United Network for Organ Sharing (UNOS) or the European Neuroendocrine Tumor Society (ENETS) criteria for LT. Kaplan-Meier survival analysis was used to calculate overall survival. RESULTS: Out of 519 patients with pan-NET, 41 patients were included. Mean follow-up time was 5.4 years. Overall survival was 9.3 years (95% Cl 6.8-11.7), and 5-year survival was 64.7% (95% CI 48.2-81.2). Patients meeting the Milan, ENETS and UNOS criteria for LT had a 5-year survival of 64.9% (95% CI 32.2-97.6), 85.7% (95% CI 59.8-100.0) and 55.4% (95% CI 26.0-84.8), respectively. CONCLUSIONS: In patients with stage IV pan-NET, grade 1 and 2, with no extra abdominal disease, 5-year survival was 64.7% (95% CI 48.2-81.2). As these survival rates exceed previously published series of LT for pan-NET, the evidence base for this treatment is very weak.