Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Low-temperature trapping of N2 reduction reaction intermediates in nitrogenase MoFe protein-CdS quantum dot complexes.

The biological reduction of N2 to ammonia requires the ATP-dependent, sequential delivery of electrons from the Fe protein to the MoFe protein of nitrogenase. It has been demonstrated that CdS nanocrystals can replace the Fe protein to deliver photoexcited electrons to the MoFe protein. Herein, light-activated electron delivery within the CdS:MoFe protein complex was achieved in the frozen state, revealing that all the electron paramagnetic resonance (EPR) active E-state intermediates in the catalytic cycle can be trapped and characterized by EPR spectroscopy. Prior to illumination, the CdS:MoFe protein complex EPR spectrum was composed of a S = 3/2 rhombic signal (g = 4.33, 3.63, and 2.01) consistent with the FeMo-cofactor in the resting state, E0. Illumination for sequential 1-h periods at 233 K under 1 atm of N2 led to a cumulative attenuation of E0 by 75%. This coincided with the appearance of S = 3/2 and S = 1/2 signals assigned to two-electron (E2) and four-electron (E4) reduced states of the FeMo-cofactor, together with additional S = 1/2 signals consistent with the formation of E6 and E8 states. Simulations of EPR spectra allowed quantification of the different E-state populations, along with mapping of these populations onto the Lowe-Thorneley kinetic scheme. The outcome of this work demonstrates that the photochemical delivery of electrons to the MoFe protein can be used to populate all of the EPR active E-state intermediates of the nitrogenase MoFe protein cycle.

The accumulation and growth of Pseudomonas aeruginosa on surfaces is modulated by surface mechanics via cyclic-di-GMP signaling.

Attachment of bacteria onto a surface, consequent signaling, and accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that surface mechanics may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of surface mechanics and modulation of accumulation in response to surface mechanics remain largely unknown. We use thin and thick hydrogels coated on glass to create composite materials with different mechanics (higher elasticity for thin composites; lower elasticity for thick composites) but with the same surface adhesivity and chemistry. The mechanical cue stemming from surface mechanics is elucidated using experiments with the opportunistic human pathogen Pseudomonas aeruginosa combined with finite-element modeling. Adhesion to thin composites results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to thick composites with identical surface chemistry. Using quantitative microscopy, we find that adhesion to thin composites also results in higher cyclic-di-GMP levels, which in turn result in lower motility and less detachment, and thus greater accumulation of bacteria on the surface than does adhesion to thick composites. Mechanics-dependent c-di-GMP production is mediated by the cell-surface-exposed protein PilY1. The biofilm lag phase, which is longer for bacterial populations on thin composites than on thick composites, is also mediated by PilY1. This study shows clear evidence that bacteria actively regulate differential accumulation on surfaces of different stiffnesses via perceiving varied mechanical stress and strain upon surface engagement.

Cryo-annealing of Photoreduced CdS Quantum Dot-Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E4(2N2H) Intermediate.

A critical step in the mechanism of N2 reduction to 2NH3 catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E4(4H)). This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H2 or going forward with N2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H2 to form the E4(2N2H) state. E4(2N2H) can relax to E4(4H) by the oxidative addition (oa) of H2 and release of N2 or can be further reduced in a series of catalytic steps to release 2NH3. If the H2 re/oa mechanism is correct, it requires that oa of H2 be associative with E4(2N2H). In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E4(2N2H). Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates. Electron paramagnetic resonance spectroscopy identified E-state signals including E2 and E4(2N2H), as well as signals suggesting the formation of E6 or E8. It is shown that in the frozen state when pN2 is much greater than pH2, the E4(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H2 to the E4(2N2H) state follows an associative reaction mechanism.

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human regulatory T cells.

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.

Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation by Pseudomonas aeruginosa.

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement.

Characterizing Autism Spectrum Disorder and Predicting Suicide Risk for Pediatric Psychiatric Emergency Services Encounters.

Individuals diagnosed with autism spectrum disorder (ASD) are at a higher risk for mental health concerns including suicidal thoughts and behaviors (STB). Limited studies have focused on suicidal risk factors that are more prevalent or unique to the population with ASD. This study sought to characterize and classify youth presenting to the psychiatric emergency department (ED) for a chief complaint of STB. The results of this study validated that a high number of patients with ASD present to the ED with STB. There were important differences in clinical characteristics to those with ASD versus those without. Clinical features that showed important impact in predicting high suicide risk in the ASD cases include elements of the mental status exam such as affect, trauma symptoms, abuse history, and auditory hallucinations. Focused attention is needed on these unique differences in ASD cases so that suicide risk level can be appropriately and promptly addressed.

The influence of electron utilization pathways on photosystem I photochemistry in Synechocystis sp. PCC 6803.

The capacity of cyanobacteria to adapt to highly dynamic photon flux and nutrient availability conditions results from controlled management and use of reducing power, and is a major contributing factor to the efficiency of photosynthesis in aquatic environments. The response to changing conditions includes modulating gene expression and protein-protein interactions that serve to adjust the use of electron flux and mechanisms that control photosynthetic electron transport (PET). In this regard, the photochemical activity of photosystem I (PSI) reaction centers can support balancing of cyclic (CEF) and linear electron flow (LEF), and the coupling of redox carriers for use by electron utilization pathways. Therefore, changes in the utilization of reducing power might be expected to result in compensating changes at PSI as a means to support balance of electron flux. To understand this functional relationship, we investigated the properties of PSI and its photochemical activity in cells that lack flavodiiron 1 catalyzed oxygen reduction activity (ORR1). In the absence of ORR1, the oxygen evolution and consumption rates declined together with a shift in the oligomeric form of PSI towards monomers. The effect of these changes on PSI energy and electron transfer properties was examined in isolated trimer and monomer fractions of PSI reaction centers. Collectively, the results demonstrate that PSI photochemistry is modulated through coordination with the depletion of electron demand in the absence of ORR1.

Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction.

The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, PN, P+, P2+ of which PN↔P+ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P+ during electron transfer, photochemical reduction of MoFe protein at 231-263 K was used to trap formation of P+ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P2+ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P+ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.

Correction: The influence of electron utilization pathways on photosystem I photochemistry in Synechocystis sp. PCC 6803.

[This corrects the article DOI: 10.1039/D2RA01295B.].

An Unsupervised Cluster Analysis of Post-COVID-19 Mental Health Outcomes and Associated Comorbidities.

The COVID-19 pandemic continues to be widespread, and little is known about mental health impacts from dealing with the disease itself. This retrospective study used a deidentified health information exchange (HIE) dataset of electronic health record data from the state of Rhode Island and characterized different subgroups of the positive COVID-19 population. Three different clustering methods were explored to identify patterns of condition groupings in this population. Increased incidence of mental health conditions was seen post-COVID-19 diagnosis, and these individuals exhibited higher prevalence of comorbidities compared to the negative control group. A self-organizing map cluster analysis showed patterns of mental health conditions in half of the clusters. One mental health cluster revealed a higher comorbidity index and higher severity of COVID-19 disease. The clinical features identified in this study motivate the need for more in-depth analysis to predict and identify individuals at high risk for developing mental illness post-COVID-19 diagnosis.

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

ABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions.

Recent Advances Clarifying the Structure and Function of Plant Apyrases (Nucleoside Triphosphate Diphosphohydrolases).

Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, structure and function of these enzymes have been published in the last half-dozen years, revealing that they carry out key functions in diverse other plants. These recent discoveries about plant apyrases include, among others, novel findings on its crystal structures, its biochemistry, its roles in plant stress responses and its induction of major changes in gene expression when its expression is suppressed or enhanced. This review will describe and discuss these recent advances and the major questions about plant apyrases that remain unanswered.

Clinical Note Section Detection Using a Hidden Markov Model of Unified Medical Language System Semantic Types.

Clinical notes are a rich source of biomedical data for natural language processing (NLP). The identification of note sections represents a first step in creating portable NLP tools. Here, a system that used a heterogeneous hidden Markov model (HMM) was designed to identify seven note sections: (1) Medical History, (2) Medications, (3) Family and Social History, (4) Physical Exam, (5) Labs and Imaging, (6) Assessment and Plan, and (7) Review of Systems. Unified Medical Language System (UMLS) concepts were identified using MetaMap, and UMLS semantic type distributions for each section type were empirically determined. The UMLS semantic type distributions were used to train the HMM for identifying clinical note sections. The system was evaluated relative to a template boundary model using manually annotated notes from the Medical Information Mart for Intensive Care III. The results show promise for an approach to segment clinical notes into sections for subsequent NLP tasks.

The Relationship Between Speech Characteristics and Motor Subtypes of Parkinson's Disease.

Purpose The aim of this study was to examine whether acoustic dysarthria characteristics align with overall motor profile in individuals with Parkinson's disease (PD). Potential speech differences between tremor-dominant and non-tremor-dominant subtypes are theoretically motivated but empirically inconclusive. Method Twenty-seven individuals with dysarthria from PD provided a contextual speech sample. Participants were grouped into non-tremor-dominant (n = 12) and tremor-dominant (n = 15) motor subtypes according to the Unified Parkinson Disease Rating Scale. Dependent speech variables included fundamental frequency range, average pause duration, cepstral peak prominence, stuttering dysfluencies, and maze dysfluencies. Results There were no significant differences between the speech of the tremor-dominant and non-tremor-dominant groups. High within-group variability existed across parameters and motor subtypes. Conclusion Speech characteristics across the areas of phonation, prosody, and fluency did not differ appreciably between PD motor subtypes.

Defining Intermediates of Nitrogenase MoFe Protein during N2 Reduction under Photochemical Electron Delivery from CdS Quantum Dots.

Coupling the nitrogenase MoFe protein to light-harvesting semiconductor nanomaterials replaces the natural electron transfer complex of Fe protein and ATP and provides low-potential photoexcited electrons for photocatalytic N2 reduction. A central question is how direct photochemical electron delivery from nanocrystals to MoFe protein is able to support the multielectron ammonia production reaction. In this study, low photon flux conditions were used to identify the initial reaction intermediates of CdS quantum dot (QD):MoFe protein nitrogenase complexes under photochemical activation using EPR. Illumination of CdS QD:MoFe protein complexes led to redox changes in the MoFe protein active site FeMo-co observed as the gradual decline in the E0 resting state intensity that was accompanied by an increase in the intensity of a new "geff = 4.5" EPR signal. The magnetic properties of the geff = 4.5 signal support assignment as a reduced S = 3/2 state, and reaction modeling was used to define it as a two-electron-reduced "E2" intermediate. Use of a MoFe protein variant, ß-188Cys, which poises the P cluster in the oxidized P+ state, demonstrated that the P cluster can function as a site of photoexcited electron delivery from CdS to MoFe protein. Overall, the results establish the initial steps for how photoexcited CdS delivers electrons into the MoFe protein during reduction of N2 to ammonia and the role of electron flux in the photochemical reaction cycle.

The toughness of porcine skin: Quantitative measurements and microstructural characterization.

An exceptional tear resistance is required of the skin to protect the body from external attacks, environmental damage, and other forms of aggression. To estimate the toughness of juvenile porcine skin, we conduct two types of experiments on pre-notched specimens, placing the tissue under shear (Mode III) by using the classical trouser test with a 25 mm long pre-notch, and opening (Mode I) with an experimental setup with the same pre-notch length. We obtain two distinct average toughness values of JIIIc≈20.4kJ/m2 and JIc=30.4kJ/m2, as a result of differences between these two modes of crack-tip loading and propagation, and collagen alignment. Digital image correlation coupled with single edge notch tests of 10 mm × 30 mm skin samples enables the mapping of the local strains around the tip of the crack. Effects of sample orientation and initial notch size ratio on the strain profile and on the net-section failure stress are discussed. The evaluation of the structure at the crack tip and regions undergoing more uniform states of deformation is conducted by ex situ transmission electron microscopy and in situ environmental scanning electron microscopy. Prior to crack propagation, the stress concentration is decreased by redistributing loads away from the crack tip, illustrated by gradual recruitment of collagen fibers ahead of the crack tip, thus delaying crack growth. After the crack has propagated, collagen fibers are substantially damaged, marked by delamination and recoil of the collagen fibrils.

Mental Health Comorbidity Analysis in Pediatric Patients with Autism Spectrum Disorder Using Rhode Island Medical Claims Data.

Identification of comorbidity subgroups linked with Autism Spectrum Disorder (ASD) could provide promising insight into learning more about this disorder. This study sought to use the Rhode Island All-Payer Claims Database to examine mental health conditions linked to ASD. Medical claims data for ASD patients and one or more mental health conditions were analyzed using descriptive statistics, association rule mining (ARM), and sequential pattern mining (SPM). The results indicated that patients with ASD have a higher proportion of mental health diagnoses than the general pediatric population. ARM and SPM methods identified patterns of comorbidities commonly seen among ASD patients. Based on the observed patterns and temporal sequences, suicidal ideation, mood disorders, anxiety, and conduct disorders may need focused attention prospectively. Understanding more about groupings of ASD patients and their comorbidity burden can help bridge gaps in knowledge and make strides toward improved outcomes for patients with ASD.

Cognitive Rehabilitation for Individuals With Parkinson's Disease: Developing and Piloting an External Aids Treatment Program.

Purpose Cognitive deficits are common in Parkinson's disease (PD) and can have a detrimental effect on daily activities. To date, most cognitive treatments have had an impairment-based focus with primary outcome measures of formal neuropsychological test scores. Few, if any, studies have focused on functional improvement or patient-centered goals. Method Three individuals with idiopathic PD participated in an 8-week pilot treatment program to train for the use of compensatory external aids to achieve personalized goals. Goal attainment scaling was the primary outcome measure, which was independently judged by multiple raters at baseline, postintervention, and 1 month posttreatment and analyzed via T-score analysis. Descriptive measures, including self-report and spouse-report rating scales of cognitive functioning, were employed. Results All 3 participants improved in the majority of their laboratory and home goals posttreatment, as measured by goal attainment scaling, and maintained gains for the majority of goals 1 month posttreatment. Conclusions This is the 1st known study to implement an external aids treatment program with patient-centered goals for individuals with cognitive deficits from PD. Positive outcomes were likely influenced by 3 factors: (a) a theoretically motivated focus on external aids; (b) a well-documented, systematic approach to instruction; and (c) the personalization of goals. Supplemental Material https://doi.org/10.23641/asha.10093493.

Coupling biology to synthetic nanomaterials for semi-artificial photosynthesis.

Biohybrid artificial photosynthesis aims to combine the advantages of biological specificity with a range of synthetic nanomaterials to create innovative semi-synthetic systems for solar-to-chemical conversion. Biological systems utilize highly efficient molecular catalysts for reduction-oxidation reactions. They can operate with minimal overpotentials while selectively channeling reductant energy into specific transformation chemistries and product forming pathways. Nanomaterials can be synthesized to have efficient light-absorption capacity and tuneability of charge separation by manipulation of surface chemistries and bulk compositions. These complementary aspects have been combined in a variety of ways, for example, where biological light-harvesting complexes function as antenna for nanoparticle catalysts or where nanoparticles function as light capture, charge separation components for coupling to chemical conversion by redox enzymes and whole cells. The synthetic diversity that is possible with biohybrids is still being explored. The progress arising from creative approaches is generating new model systems to inspire scale-up technologies and generate understanding of the fundamental mechanisms that control energy conversion at the molecular scale. These efforts are leading to discoveries of essential design principles that can enable the development of scalable artificial photosynthesis systems.