Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

Structure and ion-dependent folding of k-junctions.

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.

Biochemical and mechanistic analysis of the cleavage of branched DNA by human ANKLE1.

ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

Catalytic mechanism and pH dependence of a methyltransferase ribozyme (MTR1) from computational enzymology.

A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O6-methylguanine (O6mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy (AFE) simulations to elucidate the atomic-level solution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O6mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O6mG:N1 and rate-controlling methyl transfer (19.4 kcal·mol-1 barrier). AFE simulations predict the pKa for C10 to be 6.3, close to the experimental apparent pKa of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pKa calculations, enables us to predict an activity-pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools.

Diagnostic miRNA Signatures in Paired Tumor, Plasma, and Urine Specimens From Renal Cell Carcinoma Patients.

BACKGROUND: The incidence of patients diagnosed with renal cell carcinoma (RCC) is increasing. There are no approved biofluid biomarkers for routine diagnosis of RCC patients. This retrospective study aims to identify cell-free microRNA (cfmiR) signatures in urine samples that can be utilized as biomarkers for early diagnosis of sporadic RCC patients. METHODS: Tissue, plasma, and urine samples (n = 221) from 56 sporadic RCC patients and respective normal healthy donors were profiled for 2083 microRNAs (miRs) using the next-generation sequencing-based HTG EdgeSeq miR Whole Transcriptome Assay. DESeq2 (FC |1.2|, false discovery rate <0.05) was performed to identify differentially expressed miRs. Data from RCC tissue samples of The Cancer Genome Atlas database were used for miR validation. RESULTS: We found a 10-miR signature that distinguished RCC tissues from remote normal kidney tissue or benign kidney lesion samples. Additionally, we identified subtype-specific miRs (miR-122-5p, miR-210-3p, and miR-21-3p) and miRs specific for all RCC subtypes (miR-106b-3p, miR-629-5p, and miR-885-5p). We observed that miR-155-5p was associated with tumor size. Using The Cancer Genome Atlas data sets, we validated the miRs found in RCC tissue samples. In plasma or urine analysis, we found cfmiRs that were consistently and significantly upregulated in RCC tissue samples. A 15-cfmiR signature was proposed in urine samples of RCC patients, of which miR-1275 was consistently upregulated in tissue, plasma, and urine samples. CONCLUSIONS: This integrative study found diagnostic miRs/cfmiRs for RCC patients, which were validated using The Cancer Genome Atlas data sets. Distinctive cfmiR signatures found in urine may have clinical utility for the diagnosis of RCC.

Structure and mechanism of a methyltransferase ribozyme.

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

Malnutrition in emergency general surgery: a survey of National Emergency Laparotomy Audit Leads.

BACKGROUND: Patients who are malnourished and have emergency general surgery, such as a laparotomy, have worse outcomes than those who are not malnourished. It is paramount to identify these patients and minimise this risk. This study aimed to describe current practices in identifying malnutrition in patients undergoing a laparotomy, specifically focusing on screening, assessment, nutrition pathways and barriers encountered by clinicians. METHODS: Following piloting and validity assessment, anaesthetic and surgical National Emergency Laparotomy Audit (NELA) Leads at hospitals across England and Wales were emailed an invitation to a survey. Responses were gathered using Qualtrics. Descriptive analysis and correlation with laparotomy volume and professional role were performed in SPSSv26. University of Sheffield ethical approval was obtained (UREC 046205). The results from the survey are reported according to the CHERRIES guidelines. RESULTS: The survey was completed by 166/289 NELA Leads from 117/167 hospitals (57.4% and 70.1% response rates, respectively). Participants reported low rates of nutritional screening (42/166; 25.3%) and assessment (26/166; 15.7%) for malnutrition preoperatively. More than one third of respondents (40.1%) had no awareness of local screening tools; indeed, the Malnutrition Universal Screening Tool (MUST) was used by approximately half of respondents (56.6%). Contrary to guidelines, NELA Leads report albumin levels continue to be used to determine malnutrition risk (73.5%; 122/166). Postoperative nutrition pathways were common (71.7%; 119/166). Reported barriers to nutritional screening and assessment included a lack of time, training and education, organisational support and ownership. Participants indicated nutrition risk is inadequately identified and is an important missing data item from NELA. There was no significant correlation with hospital laparotomy volume in relation to screening or assessment for malnutrition, the use of nutritional support pathways or organisational barriers. There was interprofessional agreement across a number of domains, although some differences did exist. CONCLUSIONS: Wide variation exists in the current practice of identifying malnutrition risk in NELA patients. Barriers include a lack of time, knowledge and ownership. Nutrition pathways that encompass the preoperative phase and incorporation of nutrition data in NELA may support improvements in care.

Do conversations end when people want them to?

Do conversations end when people want them to? Surprisingly, behavioral science provides no answer to this fundamental question about the most ubiquitous of all human social activities. In two studies of 932 conversations, we asked conversants to report when they had wanted a conversation to end and to estimate when their partner (who was an intimate in Study 1 and a stranger in Study 2) had wanted it to end. Results showed that conversations almost never ended when both conversants wanted them to and rarely ended when even one conversant wanted them to and that the average discrepancy between desired and actual durations was roughly half the duration of the conversation. Conversants had little idea when their partners wanted to end and underestimated how discrepant their partners' desires were from their own. These studies suggest that ending conversations is a classic "coordination problem" that humans are unable to solve because doing so requires information that they normally keep from each other. As a result, most conversations appear to end when no one wants them to.

Lung inflammation in the pathogenesis of rheumatoid arthritis.

The primary manifestation of rheumatoid arthritis (RA) is articular disease; however, extra-articular disease can also occur. In particular, pulmonary disease is a leading cause of morbidity and mortality in individuals with RA. Herein, we will review the types, prevalence, risk factors, and potential pathophysiology of lung disease in individuals with established RA. We will also discuss the emerging understanding of potential role of the lung in the generation of RA-related autoantibodies during a period of disease development termed "pre-RA." Finally, we will discuss a research agenda outlining the next steps to improve our understanding and management of lung inflammation and lung disease throughout the natural history of RA.

Natural Antibodies Mediate Protection Against Acinetobacter baumannii Respiratory Infections.

BACKGROUND: Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pandrug-resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. METHODS: This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell-deficient (Rag2-/-) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. RESULTS: Our results showed that intranasally infected Rag2-/- mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours postinfection compared to wildtype mice. Animal pretreatment with normal mouse serum or purified antibodies from naive mice rescued Rag2-/- mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. CONCLUSIONS: Overall, our study shows that NAbs mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

Characterization of Virulence-Associated Traits in Mycoplasma penetrans Strains Acting as Likely Etiological Agents of Idiopathic Nongonococcal Urethritis.

Mycoplasma penetrans is an emerging pathogen with a reduced genome. This bacterium has only previously been cultured from individuals with chronic immunodeficiencies. Here we report the characteristics of 4 M. penetrans isolates from the urine of immunocompetent males with nongonococcal urethritis, in comparison with strain HF-2 from an immunocompromised patient. Several features exhibited distinct differences between these isolates and HF-2. Unlike HF-2, all 4 were resistant to azithromycin. They exhibited greater sialic acid-dependent binding to erythrocytes, gliding motility speed, and H2O2 production than HF-2. All new isolates produced thinner capsules than HF-2. Invasiveness varied, with some isolates being more invasive than HF-2 and some less invasive. Cytotoxicity to HeLa cells was similar to HF-2, and all strains could clear extracellular traps produced by innate immune cells. We conclude that subtle differences among M. penetrans strains may be critical for this organism to establish an infection in an otherwise healthy individual.

Absolute Configuration Determination of Chiral API Molecules by MicroED Analysis of Cocrystal Powders Formed Based on Cocrystal Propensity Prediction Calculations.

Establishing the absolute configuration of chiral active pharmaceutical ingredients (APIs) is of great importance. Single crystal X-ray diffraction (scXRD) has traditionally been the method of choice for such analysis, but scXRD requires the growth of large crystals, which can be challenging. Here, we present a method for determining absolute configuration that does not rely on the growth of large crystals. By examining microcrystals formed with chiral probes (small chiral compounds such as amino acids), absolute configuration can be unambiguously determined by microcrystal electron diffraction (MicroED). Our streamlined method employs three steps: (1) virtual screening to identify promising chiral probes, (2) experimental cocrystal screening and (3) structure determination by MicroED and absolute configuration assignment. We successfully applied this method to analyze two chiral API molecules currently on the market for which scXRD was not used to determine absolute configuration.

FCRL1 Regulates B Cell Receptor-Induced ERK Activation through GRB2.

Regulation of BCR signaling has important consequences for generating effective Ab responses to pathogens and preventing production of autoreactive B cells during development. Currently defined functions of Fc receptor-like (FCRL) 1 include positive regulation of BCR-induced calcium flux, proliferation, and Ab production; however, the mechanistic basis of FCRL1 signaling and its contributions to B cell development remain undefined. Molecular characterization of FCRL1 signaling shows phosphotyrosine-dependent associations with GRB2, GRAP, SHIP-1, and SOS1, all of which can profoundly influence MAPK signaling. In contrast with previous characterizations of FCRL1 as a strictly activating receptor, we discover a role for FCRL1 in suppressing ERK activation under homeostatic and BCR-stimulated conditions in a GRB2-dependent manner. Our analysis of B cells in Fcrl1 -/- mice shows that ERK suppression by FCRL1 is associated with a restriction in the number of cells surviving splenic maturation in vivo. The capacity of FCRL1 to modulate ERK activation presents a potential for FCRL1 to be a regulator of peripheral B cell tolerance, homeostasis, and activation.

Structure and folding of four putative kink turns identified in structured RNA species in a test of structural prediction rules.

k-Turns are widespread key architectural elements that occur in many classes of RNA molecules. We have shown previously that their folding properties (whether or not they fold into their tightly kinked structure on addition of metal ions) and conformation depend on their local sequence, and we have elucidated a series of rules for prediction of these properties from sequence. In this work, we have expanded the rules for prediction of folding properties, and then applied the full set to predict the folding and conformation of four probable k-turns we have identified amongst 224 structured RNA species found in bacterial intergenenic regions by the Breaker lab (1). We have analyzed the ion-dependence of folding of the four k-turns using fluorescence resonance energy transfer, and determined the conformation of two of them using X-ray crystallography. We find that the experimental data fully conform to both the predicted folding and conformational properties. We conclude that our folding rules are robust, and can be applied to new k-turns of unknown characteristics with confidence.

Unicorns, Rhinoceroses and Chemical Bonds.

The nascent field of computationally aided molecular design will be built around the ability to make computation useful to synthetic chemists who draw on their empirically based chemical intuition to synthesize new and useful molecules. This fact poses a dilemma, as much of existing chemical intuition is framed in the language of chemical bonds, which are pictured as possessing physical properties. Unfortunately, it has been posited that calculating these bond properties is impossible because chemical bonds do not exist. For much of the computationalchemistry community, bonds are seen as mythical-the unicorns of the chemical world. Here, we show that this is not the case. Using the same formalism and concepts that illuminated the atoms in molecules, we shine light on the bonds that connect them. The real space analogue of the chemical bond becomes the bond bundle in an extended quantum theory of atoms in molecules (QTAIM). We show that bond bundles possess all the properties typically associated with chemical bonds, including an energy and electron count. In addition, bond bundles are characterized by a number of nontraditional attributes, including, significantly, a boundary. We show, with examples drawn from solid state and molecular chemistry, that the calculated properties of bond bundles are consistent with those that nourish chemical intuition. We go further, however, and show that bond bundles provide new and quantifiable insights into the structure and properties of molecules and materials.

Tips From the Top: Do the Best Performers Really Give the Best Advice?

Everyone knows that if you want to learn how to do something, you should get advice from people who do it well. But is everyone right? In a series of studies (N = 8,693), adult participants played a game after receiving performance advice from previous participants. Although advice from the best-performing advisors was no more beneficial than advice from other advisors, participants believed that it had been-and they believed this despite the fact that they were told nothing about their advisors' performance. Why? The best performers did not give better advice, but they did give more of it, and participants apparently mistook quantity for quality. These studies suggest that performing and advising may often be unrelated skills and that in at least some domains, people may overvalue advice from top performers.

NTRK gene fusions are detected in both secretory and non-secretory breast cancers.

NTRK fusions represent a new biomarker-defined population that can be treated with TRK inhibitors. Although rare, NTRK fusions are detected across a wide range of solid tumors. Previous reports suggest that NTRK fusions are limited to the secretory subtype of breast cancer. Here we examined NTRK fusions in a large real world next-generation sequencing (NGS) dataset and confirmed secretory versus non-secretory status using H&E images. Of 23 NTRK fusion-positive cases, 11 were classified as secretory, 11 as non-secretory, and one as mixed status. The secretory subtype trended younger, was predominantly estrogen receptor (ER)-, had lower tumor mutational burden, and exhibited lower levels of genomic loss of heterozygosity. The non-secretory subtype was enriched for TP53 mutations. The secretory subtype was enriched for ETV6-NTRK3 fusions in 7 of 11 cases, and the non-secretory subtype had NTRK1 fusions in 7 of 11 cases, each with a different fusion partner. Our data suggests NTRK fusions are present in both secretory and non-secretory subtypes, and that comprehensive genomic profiling should be considered across all clinically advanced breast cancers to identify patients that could receive benefit from TRK inhibitors.

A qualitative evaluation of coproduction of research: 'If you do it properly, you will get turbulence'.

BACKGROUND: Patients and public members are increasingly involved across the different stages of the research process. Their involvement is particularly important in the conception and design of applied health research where it enables people with lived experience to influence the aims, content, focus and methods. OBJECTIVE: To evaluate the process of coproducing a mental health-related research proposal suitable for funding through a national health research funding body. METHODS: Reflections from members of the public (n = 3) and academic researchers (n = 3) were collected through semi-structured interviews. Data were thematically analysed. RESULTS: Thematic analysis identified five overarching themes: valuing the lived experience perspective; matching ambitions to the funded research process; 'Us and them': power, relationships and trust; challenges; and benefits of coproduction. CONCLUSIONS: Our findings suggest that for successful coproduction of a research funding application, an open and trusting atmosphere, where equal relationships are established and a shared common goal agreed is essential. Although relationships with research professionals were framed by trust and mutual respect for some public advisors, others felt a sense of 'us and them'. With various tensions played out through interpersonal conflict, difficult conversations and disagreements, coproduction was not a positive experience for all stakeholders involved. Among the learning was that when collaboration of this kind is constrained by time or funding, genuine, impactful coproduction can be more challenging than is generally acknowledged.

Dermal Safety of Tapinarof Cream 1%: Results From 4 Phase 1 Trials.

BACKGROUND: Tapinarof (VTAMA®; Dermavant Sciences, Inc.) is a novel, non-steroidal, topical, aryl hydrocarbon receptor agonist, FDA approved for psoriasis treatment and under investigation for atopic dermatitis treatment as a 1% cream formulation for once-daily (QD) application. OBJECTIVE: Evaluate cumulative skin irritation, sensitization, and photoallergic and phototoxic potential of tapinarof cream 1% across a range of dosing frequencies and conditions. METHODS: We conducted 4 randomized, controlled, phase 1 trials of topical tapinarof cream 1% vs vehicle or other appropriate controls in healthy adults. Cumulative skin irritation was assessed following QD application for 21 days under fully occlusive patch conditions. Contact sensitization, photoallergenicity, and phototoxicity were assessed under semi-occlusive patch conditions. The contact sensitization and photoallergenicity trials used an induction phase of repeated applications followed by a 2-week rest period and a 1-time challenge, with rechallenge if responses indicated sensitization/photosensitization; the phototoxicity trial comprised a single application. Ultraviolet A and B irradiation was used to assess photoallergenicity/toxicity. RESULTS: 376 participants were randomized across the 4 trials. In the cumulative irritation trial, tapinarof cream 1% QD was classified as having a slight potential for very mild cumulative irritation under the exaggerated test conditions of repeated dosing for 21 days. There was no evidence of sensitization, photosensitization, or phototoxicity. Tapinarof was well tolerated and there was a low discontinuation rate across all trials. CONCLUSIONS: Tapinarof cream 1% was well tolerated, non-sensitizing, non-phototoxic, and non-photoallergic, with no evidence of clinically meaningful cumulative skin irritation in 4 dermal safety trials in healthy adults. TRIAL REGISTRATION: IND 104601 J Drugs Dermatol. 2022;21(10):1084-1090. doi:10.36849/JDD.6627R1.