Tunable PhenoCycler imaging of the murine pre-clinical tumour microenvironments.

BACKGROUND: The tumour microenvironment (TME) consists of tumour-supportive immune cells, endothelial cells, and fibroblasts. PhenoCycler, a high-plex single cell spatial biology imaging platform, is used to characterize the complexity of the TME. Researchers worldwide harvest and bank tissues from mouse models which are employed to model a plethora of human disease. With the explosion of interest in spatial biology, these panoplies of archival tissues provide a valuable resource to answer new questions. Here, we describe our protocols for developing tunable PhenoCycler multiplexed imaging panels and describe our open-source data analysis pipeline. Using these protocols, we used PhenoCycler to spatially resolve the TME of 8 routinely employed pre-clinical models of lymphoma, breast cancer, and melanoma preserved as FFPE. RESULTS: Our data reveal distinct TMEs in the different cancer models that were imaged and show that cell-cell contacts differ depending on the tumour type examined. For instance, we found that the immune infiltration in a murine model of melanoma is altered in cellular organization in melanomas that become resistant to αPD-1 therapy, with depletions in a number of cell-cell interactions. CONCLUSIONS: This work presents a valuable resource study seamlessly adaptable to any field of research involving murine models. The methodology described allows researchers to address newly formed hypotheses using archival materials, bypassing the new to perform new mouse studies.

Perspective of Secondary Metabolites in Respect of Multidrug Resistance (MDR): A Review.

Aberrant and haphazard use of antibiotics has created the development of antimicrobial resistance which is a bizarre challenge for human civilization. This emerging crisis of antibiotic resistance for microbial pathogens is alarming all the nations posing a global threat to human health. It is difficult to treat bacterial infections as they develop resistance to all antimicrobial resistance. Currently used antibacterial agents inhibit a variety of essential metabolic pathways in bacteria, including macro-molecular synthesis (MMS) pathways (e.g. protein, DNA, RNA, cell wall) most often by targeting a specific enzyme or subcellular component e.g. DNA gyrase, RNA polymerase, ribosomes, transpeptidase. Despite the availability of diverse synthetic molecules, there are still many complications in managing progressive and severe antimicrobial resistance. Currently not even a single antimicrobial agent is available for which the microbes do not show resistance. Thus, the lack of efficient drug molecules for combating microbial resistance requires continuous research efforts to overcome the problem of multidrug-resistant bacteria. The phytochemicals from various plants have the potential to combat the microbial resistance produced by bacteria, fungi, protozoa and viruses without producing any side effects. This review is a concerted effort to identify some of the major active phytoconstituents from various medicinal plants which might have the potential to be used as an alternative and effective strategy to fight against microbial resistance and can promote research for the treatment of MDR.

AmpC Inhibition: An Explicit Approach against Multi-Drug Resistance (MDR).

Multi-drug resistance and its transmission is a ubiquitous health issue worldwide. The beta-lactamase AmpC resistance is a major concern among all health settings like hospitals and child care centers, etc. The clinical pipeline of the new antibiotics remains dry due to the production of AmpC beta-lactamases by the bacteria to develop resistance against antibiotics. According to the global antimicrobial resistance and use surveillance system, the rate of resistance to ciprofloxacin an antibiotic commonly used to treat urinary tract infections, varied from 8.4% to 92.9% for Escherichia coli and from 4.1% to 79.4% for Klebsiellapneumoniae in different countries. The lack of comprehensiveness within the data makes a choice problematic for the selection of appropriate ß-lactam antibiotic for the treatment of resistant microorganisms. Most experts agree it is prudent to avoid expanded-spectrum (i.e. third-generation) cephalosporins for the treatment of organisms posing the greatest risk of AmpC induction. Nonetheless, the development of specific inhibitors for the AmpC enzyme, either naturally or synthetically, is only unfolding. To date, there is no single and clinically active drug available that inhibits the AmpC enzyme and combats multidrug resistance and its transmission in individuals. The deficit of the enzyme inhibitor focused the researchers to work in the area. This present review will emphasize on the chemistry, and structure of clinically important and potent inhibitors against AmpC enzymes.

Pregnancy and Child Outcomes Following Fetal Intracranial Hemorrhage.

BACKGROUND: The prenatal and early postnatal outcomes of fetal intracranial hemorrhage (ICH) prenatally diagnosed by fetal magnetic resonance imaging (MRI) have not been well described. METHODS: A retrospective study of cases with fetal ICH diagnosed by fetal MRI at Children's National Hospital, Washington, DC, from 2012 to 2020 was conducted. Maternal characteristics, prenatal imaging, pregnancy outcome, and child developmental outcomes were recorded. Abnormal outcomes were categorized as mild for required physical/occupational therapy without other delays, moderate for intermediate multidomain developmental delays, and severe if nonambulatory, nonverbal, or intellectual disability. RESULTS: Fifty-seven cases with fetal ICH were included. The mean (S.D.) maternal age was 31.1 (6.9) years, gestational age at fetal evaluation was 28.1 (5.3) weeks, and gestational age at birth was 38.2 (1.3) weeks. Pregnancy outcomes were 75% (n = 43) live birth, 14% (n = 8) termination of pregnancy, and 11% (n = 6) intrauterine demise (IUD). Live births decreased from 90% to 33% and IUD increased 10% to 22% when comparing unilateral intraventricular hemorrhage with more extensive hemorrhages. Among the 37 live-born infants with clinical follow-up to age 1.8 (1.6) years, neurodevelopmental outcome was normal in 57%, mildly abnormal in 24%, moderately abnormal in 14%, and severely abnormal in 5%. In five cases, an etiology was identified: two had placental pathologies, two had genetic findings (fetal neonatal alloimmune thrombocytopenia and COL4A1 mutation), and one had congenital cytomegalovirus infection. CONCLUSION: Perinatal and early child outcomes following fetal ICH have a wide spectrum of outcomes. Fetal MRI description of ICH location may aid in pregnancy and postnatal outcome prediction.

Social Media Use by Residents and Faculty in Otolaryngology Training Programs.

OBJECTIVES: Despite the growth of social media in healthcare, the appropriateness of online friendships between otolaryngological residents and attendings is poorly defined in the current literature. This issue is of growing importance, particularly as residency programs increasingly utilize social media as a means of connecting with and evaluating applicants due to limited in-person experiences during the COVID-19 pandemic. Our objective was to better understand the prevalence of and concerns surrounding social media use between residents and faculty. METHODS: This study sent out 2 surveys in 2017 to all United States Otolaryngology residency program directors to disperse to their residents and attendings, respectively. RESULTS: We received a response from 72 residents and 98 attendings. Our findings show that social media is commonly used by both residents and attendings, and most residents have at least 1 online friendship with an attending. Resident and attending opinions diverge on topics such as appropriateness of use, privacy settings, and professionalism. CONCLUSIONS: We call on residency programs to delineate a transparent social media policy so applicant expectations on social media are clear.

Lipid-mediated antimicrobial resistance: a phantom menace or a new hope?

The proposition of a post-antimicrobial era is all the more realistic with the continued rise of antimicrobial resistance. The development of new antimicrobials is failing to counter the ever-increasing rates of bacterial antimicrobial resistance. This necessitates novel antimicrobials and drug targets. The bacterial cell membrane is an essential and highly conserved cellular component in bacteria and acts as the primary barrier for entry of antimicrobials into the cell. Although previously under-exploited as an antimicrobial target, the bacterial cell membrane is attractive for the development of novel antimicrobials due to its importance in pathogen viability. Bacterial cell membranes are diverse assemblies of macromolecules built around a central lipid bilayer core. This lipid bilayer governs the overall membrane biophysical properties and function of its membrane-embedded proteins. This mini-review will outline the mechanisms by which the bacterial membrane causes and controls resistance, with a focus on alterations in the membrane lipid composition, chemical modification of constituent lipids, and the efflux of antimicrobials by membrane-embedded efflux systems. Thorough insight into the interplay between membrane-active antimicrobials and lipid-mediated resistance is needed to enable the rational development of new antimicrobials. In particular, the union of computational approaches and experimental techniques for the development of innovative and efficacious membrane-active antimicrobials is explored.

A Unique Sequence Is Essential for Efficient Multidrug Efflux Function of the MtrD Protein of Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the resistance-nodulation-cell division (RND) superfamily of efflux transporters, the MtrD membrane protein of N. gonorrhoeae provides resistance to a broad range of antimicrobial compounds. A unique feature of MtrD is an 11-residue sequence (from N917 to P927 [N917-P927]) that connects transmembrane helices (TMS) 9 and 10; this sequence is not present in homologous RND proteins. This study explores the structural and functional roles of the N917-P927 region by means of mutant analysis and molecular dynamics simulations. We show that N917-P927 plays a key role in modulating substrate access to the binding cleft and influences the overall orientation of the protein within the inner membrane necessary for optimal functioning. Removal of N917-P927 significantly reduced MtrD-mediated resistance to a range of antimicrobials and mutations of three single amino acids impacted MtrD-mediated multidrug resistance. Furthermore, molecular dynamics simulations showed deletion of N917-P927 in MtrD may dysregulate access of the substrate to the binding cleft and closure of the substrate-binding pocket during the transport cycle. These findings indicate that N917-P927 is a key region for interacting with the inner membrane, conceivably influencing substrate capture from the membrane-periplasm interface and thus is essential for full multidrug resistance capacity of MtrD. IMPORTANCE The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance. This study demonstrates that a unique region of the MtrD protein that connects TMS 9 and TMS 10 forms a structure that may interact with the inner membrane positioning TMS 9 and stabilizing the protein facilitating substrate capture from the inner membrane-periplasm interface. Analysis of mutants of this region identified that it was essential for MtrD-mediated multidrug resistance. Characterization of the structure and function of this unique local region of MtrD has implications for drug efflux mechanisms used by related proteins and is important knowledge for development of antibiotics that bypass efflux.

Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism.

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.

Feel the Burn! Fireworks-related Otolaryngologic Trauma.

OBJECTIVES: Fireworks are used commonly for celebrations in the United States, but can lead to severe injury to the head and neck. We aim to assess the incidence, types, and mechanisms of head and neck injuries associated with fireworks use from 2010 to 2019. METHODS: A retrospective cross-sectional study, using data from the National Electronic Injury Surveillance System, of individuals presenting to United States Emergency Departments with head and neck injuries caused by fireworks and flares from 2010 to 2019. Incidence, types, and mechanisms of injury related to fireworks use in the US population were assessed. RESULTS: A total of 541 patients (349 [64.5%] male, and 294 [54%] under 18 years of age) presented to emergency departments with fireworks-related head and neck injuries; the estimated national total was 20 584 patients (13 279 male, 9170 white, and 11 186 under 18 years of age). The most common injury diagnoses were burns (44.7% of injuries), laceration/avulsion/penetrating trauma (21.1%), and otologic injury (15.2%), which included hearing loss, otalgia, tinnitus, unspecified acoustic trauma, and tympanic membrane perforation. The remaining 19% of injuries were a mix, including contusion, abrasion, hematoma, fracture, and closed head injury. Associations between fireworks type and injury diagnosis (chi-square P < .001), as well as fireworks type by age group (chi-square P < .001) were found. Similarly, associations were found between age groups and injury diagnoses (chi-square P < .001); these included children 5 years and younger and adults older than 30 years. CONCLUSIONS: Fireworks-related head and neck injuries are more likely to occur in young, white, and male individuals. Burns are the most common injury, while otologic injury is a significant contributor. Annual rates of fireworks-related head and neck injuries have not changed or improved significantly in the United States in the past decade, suggesting efforts to identify and prevent these injuries are insufficient.

Nascent peptide assists the ribosome in recognizing chemically distinct small molecules.

Regulation of gene expression in response to the changing environment is critical for cell survival. For instance, binding of macrolide antibiotics to the ribosome promotes translation arrest at the leader open reading frames ermCL and ermBL, which is necessary for inducing the antibiotic resistance genes ermC and ermB. Cladinose-containing macrolides such as erythromycin (ERY), but not ketolides such as telithromycin (TEL), arrest translation of ermCL, whereas either ERY or TEL stall ermBL translation. How the ribosome distinguishes between chemically similar small molecules is unknown. We show that single amino acid changes in the leader peptide switch the specificity of recognition of distinct molecules, triggering gene activation in response to ERY alone, to TEL alone or to both antibiotics or preventing stalling altogether. Thus, the ribosomal response to chemical signals can be modulated by minute changes in the nascent peptide, suggesting that protein sequences could have been optimized for rendering translation sensitive to environmental cues.