Extracting thermodynamic properties from van 't Hoff plots with emphasis on temperature-sensing ion channels.

Transient receptor potential (TRP) ion channels are among the most well-studied classes of temperature-sensing molecules. Yet, the molecular mechanism and thermodynamic basis for the temperature sensitivity of TRP channels remains to this day poorly understood. One hypothesis is that the temperature-sensing mechanism can simply be described by a difference in heat capacity between the closed and open channel states. While such a two-state model may be simplistic it nonetheless has descriptive value, in the sense that it can be used to compare overall temperature sensitivity between different channels and mutants. Here, we introduce a mathematical framework based on the two-state model to reliably extract temperature-dependent thermodynamic potentials and heat capacities from measurements of equilibrium constants at different temperatures. Our framework is implemented in an open-source data analysis package that provides a straightforward way to fit both linear and nonlinear van 't Hoff plots, thus avoiding some of the previous, potentially erroneous, assumptions when extracting thermodynamic variables from TRP channel electrophysiology data.

Bacteriophages avoid autoimmunity from cognate immune systems as an intrinsic part of their life cycles.

Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned off during prophage induction, balancing immunity and autoimmunity. Our results show that the fine regulation of these processes is essential for the correct development of prophages' life cycle.

High-harmonic spectroscopy of impulsively aligned 1,3-cyclohexadiene: Signatures of attosecond charge migration.

High-harmonic spectroscopy is an all-optical technique with inherent attosecond temporal resolution that has been successfully employed to reconstruct charge migration, electron-tunneling dynamics, and conical-intersection dynamics. Here, we demonstrate the extension of two key components of high-harmonic spectroscopy, i.e., impulsive alignment and measurements with multiple driving wavelengths to 1,3-cyclohexadiene and benzene. In the case of 1,3-cyclohexadiene, we find that the temporal sequence of maximal and minimal emitted high-harmonic intensities as a function of the delay between the alignment and probe pulses inverts between 25 and 30 eV and again between 35 and 40 eV when an 800-nm driver is used, but no inversions are observed with a 1420-nm driver. This observation is explained by the wavelength-dependent interference of emission from multiple molecular orbitals (HOMO to HOMO-3), as demonstrated by calculations based on the weak-field asymptotic theory and accurate photorecombination matrix elements. These results indicate that attosecond charge migration takes place in the 1,3-cyclohexadiene cation and can potentially be reconstructed with the help of additional measurements. Our experiments also demonstrate a pathway toward studying photochemical reactions in the molecular frame of 1,3-cyclohexadiene.

[Feasibility of clinical examination scenarios under pandemic conditions]. / Möglichkeit von Prüfungsszenarien unter Pandemiebedingungen.

BACKGROUND: Due to the COVID-19 pandemic, contact restrictions occurred worldwide, which affected medical schools as well. It was not possible to hold classroom lectures. Teaching contents had to be converted to a digital curriculum within a very short time. Conditions for assessments posed an even greater challenge. For example, solutions had to be found for objective structured clinical examinations (OSCE), which were explicitly forbidden in some German states. The aim of this study was to evaluate the feasibility of an OSCE under pandemic conditions. MATERIALS AND METHODS: At the end of the 2020 summer semester, 170 students completed a combined otolaryngology and ophthalmology OSCE. Examinations were held in small groups over the course of 5 days and complied with strict hygiene regulations. The ophthalmology exam was conducted face to face, and the ENT OSCE virtually. Students were asked to rate the OSCE afterwards. RESULTS: Between 106 and 118 of the students answered the questions. Comparing the face-to-face OSCE with the virtual OSCE, about 49% preferred the face-to-face OSCE and 17% preferred the virtual OSCE; 34% found both variants equally good. Overall, the combination of an ENT and ophthalmology OSCE was rated as positive. CONCLUSION: It is possible to hold an OSCE even under pandemic conditions. For optimal preparation of the students, among other things, it is necessary to transform teaching contents to a digital curriculum. The combination of an ENT and ophthalmology OSCE was positively evaluated by the students, although the face-to-face OSCE was preferred. The overall high satisfaction of the students confirms the feasibility of a virtual examination with detailed and well-planned preparation.

DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2.

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles.

The CRISPR effector Cam1 mediates membrane depolarization for phage defence.

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

Genetic prediction of 33 blood group phenotypes using an existing genotype dataset.

BACKGROUND: Accurate blood type data are essential for blood bank management, but due to costs, few of 43 blood group systems are routinely determined in Danish blood banks. However, a more comprehensive dataset of blood types is useful in scenarios such as rare blood type allocation. We aimed to investigate the viability and accuracy of predicting blood types by leveraging an existing dataset of imputed genotypes for two cohorts of approximately 90,000 each (Danish Blood Donor Study and Copenhagen Biobank) and present a more comprehensive overview of blood types for our Danish donor cohort. STUDY DESIGN AND METHODS: Blood types were predicted from genome array data using known variant determinants. Prediction accuracy was confirmed by comparing with preexisting serological blood types. The Vel blood group was used to test the viability of using genetic prediction to narrow down the list of candidate donors with rare blood types. RESULTS: Predicted phenotypes showed a high balanced accuracy >99.5% in most cases: A, B, C/c, Coa /Cob , Doa /Dob , E/e, Jka /Jkb , Kna /Knb , Kpa /Kpb , M/N, S/s, Sda , Se, and Yta /Ytb , while some performed slightly worse: Fya /Fyb , K/k, Lua /Lub , and Vel ~99%-98% and CW and P1 ~96%. Genetic prediction identified 70 potential Vel negatives in our cohort, 64 of whom were confirmed correct using polymerase chain reaction (negative predictive value: 91.5%). DISCUSSION: High genetic prediction accuracy in most blood groups demonstrated the viability of generating blood types using preexisting genotype data at no cost and successfully narrowed the pool of potential individuals with the rare Vel-negative phenotype from 180,000 to 70.

An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCCmec Cassette.

CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCCmec) type V(5C2&5), conferring ß-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCCmec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus, CRISPR-Cas is rare, but when present, it is located within the SCCmec element, which encodes resistance to methicillin and other ß-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCCmec elements in different species of non-S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus. Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.

Wing morphology of a damselfly exhibits local variation in response to forest fragmentation.

Environmental differences can lead to morphologically different subpopulations. The scale of the mosaic of morphologies should help shed light on the nature of the mechanisms at work. Previous work has shown that jewelwing damselflies have different wing sizes in different types of habitat. Our aim was to (1) describe the relationship between damselfly wing lengths and a gradient of forest fragmentation and (2) determine the spatial scale at which these morphological differences occur. We hypothesized that local adaptation would lead to differences in wing morphology over short distances. We herein test one of the several predictions that would need to be met to support this hypothesis: that wing morphology would show spatial autocorrelation at relatively short distances. We further predicted that the wing morphology would correlate to forest fragmentation. We collected jewelwing damselflies from across Indiana, USA, in habitats across a gradient of forest fragmentation. We examined the link between forest edge density and wing length using three biologically relevant landscape sizes. We then examined the distance to which wing length variation was autocorrelated using Moran's I. We found positive linear or unimodal relationships between wing length and edge density, in both males and females, at all three landscape scales. Spatial autocorrelation in wing length indicated that variation in wing length was autocorrelated at short distances, out to 1-5 km. Our findings uphold one of the predictions stemming from the hypothesis that adaptations to local environments-habitat fragmentation here-can occur at relatively fine spatial scales.

Anisotropic Friction in a Ligand-Protein Complex.

The effect of an externally applied directional force on molecular friction is so far poorly understood. Here, we study the force-driven dissociation of the ligand-protein complex biotin-streptavidin and identify anisotropic friction as a not yet described type of molecular friction. Using AFM-based stereographic single molecule force spectroscopy and targeted molecular dynamics simulations, we find that the rupture force and friction for biotin-streptavidin vary with the pulling angle. This observation holds true for friction extracted from Kramers' rate expression and by dissipation-corrected targeted molecular dynamics simulations based on Jarzynski's identity. We rule out ligand solvation and protein-internal friction as sources of the angle-dependent friction. Instead, we observe a heterogeneity in free energy barriers along an experimentally uncontrolled orientation parameter, which increases the rupture force variance and therefore the overall friction. We anticipate that anisotropic friction needs to be accounted for in a complete understanding of friction in biomolecular dynamics and anisotropic mechanical environments.

Urothelial Carcinoma of the Bladder With Primary Metastasis to the Brain: A Case Report and Literature Review.

Brain metastases are the most common type of brain tumor in adults, commonly arising from primary tumor sites of the lung, breast, skin (melanoma), colon, and kidney. Isolated central nervous system (CNS) metastasis arising from urothelial carcinoma (UC) is a rare presentation yielding a poor prognosis. A 71-year-old male patient with a history of urothelial carcinoma, treated one year prior with partial cystectomy and adjuvant gemcitabine and cisplatin (GC) therapy, presented with worsening neurological symptoms, including progressively worsening dizziness, shuffling gait, drifting, expressive aphasia, and confusion. MRI revealed a left frontal 4.0 x 3.6 cm brightly contrast-enhancing tumor with possible hemorrhage, extensive vasogenic edema, and moderate mass effect. An additional smaller right cerebellar lesion was also noted. Outpatient CT of his chest, abdomen, and pelvis revealed no evidence of other malignant sites. He ultimately underwent a left craniotomy with a total resection of his left frontal mass. Pathological examination revealed a urothelial primary. Post-operative MRI revealed complete resection of the left frontal mass and the patient was discharged with no neurologic deficits on exam. In many cases, brain metastases may present years later following initial therapy of UC as the CNS may act as a sanctuary site during systemic chemotherapy. Chemotherapeutics such as gemcitabine with better penetration of the blood-brain barrier may be beneficial in delaying the onset of these metastases.

Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors.

Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.

Angle-dependent strength of a single chemical bond by stereographic force spectroscopy.

A wealth of chemical bonds and polymers have been studied with single-molecule force spectroscopy, usually by applying a force perpendicular to the anchoring surface. However, the direction-dependence of the bond strength lacks fundamental understanding. Here we establish stereographic force spectroscopy to study the single-bond strength for various pulling angles. Surprisingly, we find that the apparent bond strength increases with increasing pulling angle relative to the anchoring surface normal, indicating a sturdy mechanical anisotropy of a chemical bond. This finding can be rationalized by a fixed pathway for the rupture of the bond, resulting in an effective projection of the applied pulling force onto a nearly fixed rupture direction. Our study is fundamental for the molecular understanding of the role of the direction of force application in molecular adhesion and friction. It is also a prerequisite for the nanoscale tailoring of the anisotropic strength of bottom-up designed materials.

Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic.

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.157T>C, p.Ser53Pro missense variant. Although absent from reference databases, p.Ser53Pro occurred with a minor allele frequency of 0.034 in their Inuit ancestry. The serine to proline substitution prevented cell surface expression of IFNAR2 protein, small amounts of which persisted intracellularly in an aberrantly glycosylated state. Cells exclusively expressing the p.Ser53Pro variant lacked responses to recombinant IFN-I and displayed heightened vulnerability to multiple viruses in vitro-a phenotype rescued by wild-type IFNAR2 complementation. This novel form of autosomal recessive IFNAR2 deficiency reinforces the essential role of IFN-I in viral immunity. Further studies are warranted to assess the need for population screening.

Drug Levels Associated With Optimal Discrimination Between Remission and Nonremission and Comparison of Antibody Assays During First Year of Stable Infliximab Maintenance Therapy in Inflammatory Bowel Disease.

BACKGROUND: To implement therapeutic drug monitoring-based strategies for infliximab (IFX) in inflammatory bowel disease, the authors assessed IFX levels for optimal discrimination between remission and nonremission and compared assays for anti-IFX antibodies (Abs). METHODS: The retrospective cohort comprised 163 bionaive patients with inflammatory bowel disease who received stable IFX maintenance therapy (5 mg/kg every 8 weeks [q8w]) for 1 year. The clinical and biochemical remission status was assessed at all infusions (weeks 14-22-30-38-46-54), and IFX and anti-IFX Abs were estimated using a time-resolved fluorometric assay (n = 690; 88% of infusions). Samples positive for anti-IFX Abs or IFX levels < limit of detection (n = 102) were analyzed by 2 binding assays [enzyme-linked immunosorbent assay (ELISA)] and functional reporter gene assay/drug-tolerant enzyme immunoassay. RESULTS: At all assessed time points, IFX levels were more than double in patients presenting clinical or biochemical remission. An IFX concentration of 4.5 mcg/mL was associated with clinical remission during the entire first year of therapy [sensitivity 54% (49-59), specificity 73% (67-78), AUCROC 0.65 (0.60-0.69), P < 0.0001]; these values were comparable with biochemical remission. Exploratory assessments for endoscopic remission (n = 131) were performed at the discretion of the treating physician. Anti-IFX Abs were associated with undetectable IFX and treatment failure [OR 2.9 (1.4-6.0), P < 0.01], irrespective of persistency or transiency. All performed assays detected anti-IFX Abs were picked up by all assays in ∼2/3 of samples. Binding assays demonstrated a higher sensitivity to anti-IFX Abs. CONCLUSIONS: IFX at ∼5 mcg/mL was associated with clinical and biochemical remission during the first year of maintenance therapy. During this phase of therapy, standard binding assays are appropriate for therapeutic drug monitoring.

Postpartum Disseminated Herpes Simplex Virus Type 1 Infection With Hemophagocytic Lymphohistiocytosis and Fulminant Neonatal Herpes Infection.

The present study describes a 19-year-old woman with systemic herpes simplex virus (HSV)-1 infection and hemophagocytic lymphohistiocytosis (HLH) postpartum, and a fatal course of neonatal herpesvirus infection. Functional investigation of cells from the mother demonstrated significantly impaired induction of antiviral interferons and cytokines in the context of normal activation of the transcription factors NF-κB and IRF3. Whole-exome sequencing did not reveal any functionally validated genetic variants. We suggest that the functionally impaired antiviral responses, potentially caused by a variant in CASP8 or other variants in noncoding regions of the genome, contributed to the unusually severe disease course observed in two generations.

Apremilast effectively inhibits TNFα-induced vascular inflammation in human endothelial cells.

BACKGROUND: Patients with chronic inflammatory diseases (e.g. psoriasis and rheumatoid arthritis) are at increased risk for the development of atherosclerosis and cardiovascular diseases (CVD). Previous studies have suggested that phosphodiesterase 4 (PDE4) inhibitors possess anti-inflammatory properties. OBJECTIVES: Here we examined the effect of the PDE4 inhibitor apremilast, a well-established anti-psoriatic drug, on pro-inflammatory responses in TNFα-activated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) were treated with tumour necrosis factor-α (TNFα) in the presence or absence of apremilast. Expression levels of pro-inflammatory cytokines, chemokines and adhesion molecules were assessed by ELISA, western blot and RT-PCR. Effects of apremilast on adhesion and transendothelial migration (TEM) of THP-1 monocytic cells were analysed in transwell assays. RESULTS: Apremilast suppressed TNFα-induced expression and secretion of important endothelial and monocytic pro-inflammatory factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF), C-X-C motif chemokine ligand 10 (CXCL10), chemokine (C-C motif) ligand 2 (CCL2), vascular cell adhesion molecule 1 (VCAM-1), E-selectin and matrix metalloproteinase-9 (MMP9). Functionally, apremilast reduced adhesion of THP-1 cells to activated HUVECs and TEM in response to TNFα. Mechanistically, apremilast suppressed activation of nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPK) signalling in activated HUVECs. Furthermore, inhibition of p38, C-Jun-N-terminale Kinase (JNK) and NFκB in activated HUVECs decreased expression of GM-CSF, VCAM-1 and E-selectin. Additionally, apremilast decreased IL-17A-induced secretion of IL-6 and CCL2. CONCLUSIONS: We demonstrate that apremilast has distinct anti-inflammatory effects in activated HUVECs, indicating that apremilast could have the therapeutic potential to prevent higher risk for CVD in patients with chronic inflammatory diseases.

Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes.

CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR-cas locus is a recurrent mechanism to counteract immunity.

Latent effects of early-life methylmercury exposure on motor function in Drosophila.

The developmental toxicant, methylmercury (MeHg), can elicit motor deficits that last well into adulthood. Recent studies using Drosophila showed that the developing musculature is sensitive to high doses of MeHg, where a larval feeding paradigm resulted in compromised myotendinous junction (MTJ) formation during development, by a mechanism involving the NG2 homologue, kon-tiki (kon). Low-dose exposures to MeHg that do not produce muscle pathology during development, nevertheless result in impaired flight behavior later in adult life. The present study evaluated the potential for relatively low-dose exposure to produce latent adult muscle pathology and motor impairments, as assayed by climbing and flight, as well as to evaluate molecular mechanisms that may contribute to motor deficits. Wildtype larvae were fed 0, 2, 2.5, or 5 µM MeHg laden food until eclosion. The effect of 5 µM MeHg on MTJ-related gene expression during pupal development was assessed via quantitative RT-qPCR analysis. Upon eclosion, adults were transferred to standard food bottles for 4, 11, or 30 days prior to motor testing. Survivorship (%) was determined from a subset of 200 flies per treatment. Average climbing speed (cm/s) was quantified 4-days post-eclosion (PE). Flight ability was assayed 11- or 30-days PE by measuring landing height (cm) of flies dropped into an adhesive-lined vertical column. In parallel, total body mercury was measured to estimate the influence of residual MeHg at the time of motor testing. Muscle morphology was assessed using immuno-fluorescence microscopy. Exposure to 5uM MeHg significantly reduced climbing speed, and flight ability 4 and 11 - days PE, respectively. While age-related flight deficits were seen in each sex, flight deficits due to MeHg persisted to 30-day PE timepoints exclusively in males. Expression of kon was upregulated across the window of pupal development essential to establishing adult MTJ. However, experimentally restricting the induction of comparable levels of kon to muscle during the same periods did not recapitulate the flight deficits, indicating that muscle-specific induction of kon alone is not sufficient to contribute to latent flight impairments. Adult flight muscle morphology of 11-day PE flies treated with 5 µM MeHg was indistinct from controls, implying muscle structure is not grossly perturbed to impair flight. Collectively, the current data suggest that developmental exposure to 5 µM MeHg reduces flight ability in each sex at 11 day-PE and that latent deficits at 30-day PE are male-specific. It remains to be determined whether the developing MTJ of Drosophila is a sensitive target of MeHg, and whether or not kon acts in conjunction with additional MTJ factors to constitute a MeHg target.

Neuroligin-1 Is a Mediator of Methylmercury Neuromuscular Toxicity.

Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. We investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, whereas a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg1 and nrx1 revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20-36-h window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. We identified Nlg1 as a muscle-specific, NMJ structural component that can mediate MeHg neuromuscular toxicity resulting from early life exposure.