bluuebunny/crossref_metadata_2025 huggingface dataset:
Provider Experience With the Use of Ketamine for Refractory Status Epilepticus
Authors: Tantillo Gabriela , Davis Nicole , Granstein Justin , Yoo Ji Yeoun , Agarwal Parul , Reilly Kaitlin , Reynolds Alexandra , Kayal Gina , Liang John , Jetté Nathalie
Published: March 2024

Objective

Refractory status epilepticus (RSE) treated with anesthetic agents can be associated with complications including respiratory depression and hypotension. Ketamine is an emerging RSE treatment, but optimal dosing and timing are unknown. We studied provider attitudes and practices regarding the use of ketamine for RSE.

Methods

A literature review informed the creation of the survey, developed by professionals in epilepsy, pharmacy, and neurocritical care. The survey was distributed to members of the Critical Care EEG Monitoring and Research Consortium, Neurocritical Care Society, American Academy of Neurology Synapse community, American Epilepsy Society, and the Canadian League Against Epilepsy. Descriptive statistics were calculated.

Results

There were 109 respondents. First-line agents for RSE were midazolam (53%), propofol (42%), pentobarbital (2%), and ketamine (1%). Reasons for ketamine use included failure of midazolam/propofol to control seizures (81%) or hypotension on another anesthetic (35%). Perceived contraindications included hypertension (37%), elevated intracranial pressure (24%), and heart failure (18%). Perceived benefits included decreased use of vasopressors (53%) and more rapid RSE control when used adjunctively (49%). Routine ketamine users often treated more than 10 RSE cases per year, worked as intensivists or at academic institutions. Of the respondents, 59% found ketamine useful for RSE and 94% were interested in learning more about its use.

Conclusions

Although most participants found ketamine helpful for RSE, it is mainly used as a second-line agent adjunctively with midazolam or propofol. Perceived ketamine benefits included decreased need for hemodynamic support and more rapid seizure control when used in conjunction with other anesthetics. Perceived contraindications centered on cardiac and intracranial pressure concerns.
Association of Anticholinergic Drug Burden With Cognitive and Functional Decline Over Time in Dementia With Lewy Bodies: 1-Year Follow-Up Study
Authors: Ozsurekci Cemile , Satis Neslihan Kayahan , Demircan Sultan Keskin , Naharci Mehmet Ilkin
Published: March 2024

Background

The purpose of this study was to investigate the relationship between anticholinergic burden (ACB), and cognitive and functional alterations in patients with dementia of Lewy bodies (DLB) during a 1-year follow-up period.

Methods

This cohort study included patients diagnosed with DLB admitted to a tertiary geriatric outpatient clinic. Cognition, functional performance, and nutritional status were assessed at baseline, 6 months, and 12 months during the follow-up period. The ACB was evaluated, and participants were grouped as ACB ≥1 and ACB=0.

Results

A total of 112 patients with DLB (mean age, 79.3 ± 6.8 years; 50.9% female) were included. The mean number of medications was 5.1 ± 4, 56.9% of participants had polypharmacy, and 55.2% had an anticholinergic drug burden. Individuals with ACB ≥1 had lower instrumental activities of daily living (IADL) scores at baseline than those with ACB=0 (P=0.014). The Barthel index and Lawton-Brody IADL scores significantly decreased in the ACB ≥1 group on repetitive measurements over time, whereas only the Lawton-Brody IADL scores worsened in the ACB=0 group (allP<0.001). There were no significant differences in cognitive scores and Mini-Mental State Examination subdomains between the groups. The dependent variable repetitive test revealed a significant deterioration in the orientation subdomain in the ACB ≥1 group over time (P=0.001). Multivariable regression models showed no significant effect of ACB score on cognitive and functional impairment.

Conclusion

Our study provides evidence that the use of anticholinergic drugs in this vulnerable population may potentially increase the morbidity by adversely affecting functional status and cognitive orientation.
Development of selective isolation media for detecting Actinomyces and Schaalia genera from oral specimens containing indigenous bacteria
Authors: Aoki Sadao , Yahara Hiroko , Nakao Ryoma , Tsuzukibashi Osamu , Yahara Koji
Published: March 2024

To isolate specific bacteria from samples constituting the microbiota, it is essential to employ selective media that suppress the growth of resident bacteria other than specific target bacteria. Selective media for clinically important Actinomyces (including Schaalia, which was previously taxonomically classified as part of the Actinomyces genus) have been limited because they have been designed for a limited range of species within the genus and require ingredients which are difficult to prepare and handle. This study aimed to develop a selective medium (referred to as Actinomyces and Schaalia Selective Medium (ASSM)) for the isolation of a broad range of Actinomyces and Schaalia species from samples mixed with resident bacteria.  The composition of ASSM includes yeast extract, agar, brain heart infusion (BHI), levofloxacin (LVFX), fosfomycin (FOM), colistin (CL), and metronidazole (MNZ). Evaluation of the medium using 24 swab samples serially collected from the roots of the teeth of a healthy individual for whom metagenome sequencing data of a saliva sample are publicly available revealed that ASSM adjusted to concentrations of LVFX 0.5 mg/L, FOM 5 mg/L, CL 1 mg/L, and MNZ 2 mg/L and cultured anaerobically at 35°C for 7 days enabled the isolation of Actinomyces species from 37.5% of the samples. The inclusion of CL and MNZ in ASSM can also be useful for samples suspected of harbouring other bacterial species.  The selective isolation medium is expected to contribute to studies investigating the relationship between these bacteria and their pathogenesis or disease.
Effect of 10 days of unilateral lower limb suspension on knee extensors neuromuscular function and spinal excitability
Authors: Lebesque Loic , Narici Marco , Martin Alain , De Vito Giuseppe , Sarto Fabio , Scaglioni Gil
Published: March 2024

The reduction in mechanical loading applied on the lower limb has numerous detrimental consequences on neuromuscular function. The current study aimed to investigate the changes in knee extensors strength and spinal excitability induced by unilateral lower limb suspension (ULLS), providing new insights into the neuromuscular adaptations to muscle hypoactivity. Ten young healthy males (19-28 years old) underwent 10 days of ULLS to simulate muscle disuse. Modulation by unloading of knee extensors function (muscle morphology and strength, activation capacity and contractile properties) and spinal reflexes were explored before and after the ULLS. The knee extensors anatomical cross-sectional area (-4%, p = 0.007), maximal strength (-27%, p < 0.001) and central activation ratio (-3%, p = 0.006) were reduced after 10 days of ULLS. Vastus medialis H-reflex amplitude was enhanced both at rest (+33%, p = 0.038) and during a low-intensity contraction set at 10% of maximal strength (+103%, p = 0.038). No changes in muscle contractility and nerve conduction velocity were observed after the ULLS. The present study suggests that neural impairments mainly contribute to the decrease in knee extensors strength induced by short-term ULLS. The decrease in muscle activation after a short period of ULLS was accompanied by an increase in spinal excitability. However, the latter adaptation did not counterbalance the reduction in activation capacity and thus in maximal strength resulting from ULLS. These adaptations to short-term ULLS should be considered when aiming at improving the neuromuscular function of people experiencing muscle hypoactivity.
PVN-mPFC OT projections modulates pup-directed pup care or attacking in virgin mandarin voles
Authors: Li Lu , Li Yin , Huang Caihong , Hou Wenjuan , Lv Zijian , Zhang Lizi , Qu Yishan , Sun Yahan , Huang Kaizhe , Han Xiao , He Zhixiong , Tai Fadao
Published: March 2024

In many species, adult animals may exhibit caregiving or aggression towards conspecific offspring. The neural mechanisms underlying the infanticide and pup care remain poorly understood. Here, using monogamous virgin mandarin voles (Microtus mandarinus) that may exhibit pup care or infanticide, we found that more oxytocin (OT) neurons in the paraventricular nucleus (PVN) were activated during pup caring than infanticide. Optogenetic activation of OT neurons in the PVN facilitated pup-caring in male and female mandarin voles. In infanticide voles, optogenetic activation of PVN OT cells prolonged latency to approach and attack pups, whereas inhibition of these OT neurons facilitated approach and infanticide. In addition, OT release in the medial prefrontal cortex (mPFC) in pup-care voles increased upon approaching and retrieving pups, and decreased in infanticide voles upon attacking pups. Optogenetic activation of PVN OT neuron projections to the mPFC shortened the latency to approach and retrieve pups and facilitated the initiation of pup care, whereas inhibition of these projections had little effect. For pup-care females, neither activation nor inhibition of the fibers affected their behavior towards pups. In infanticide male and female voles, optogenetic activation of PVN-mPFC OT projection fibers prolonged the latency to approach and attack pups and suppressed the initiation of infanticide, whereas inhibition of these projections promoted approach and infanticide. Finally, we found that intraperitoneal injection of OT promoted pup care and inhibited infanticide behavior. It is suggested that the OT system, especially PVN OT neurons projecting to mPFC, modulates pup-directed behaviors and OT can be used to treat abnormal behavioral responses associated with some psychological diseases such as depression and psychosis.
Air-liquid interface exposure of A549 human lung cells to characterize the hazard potential of a gaseous bio-hybrid fuel blend
Authors: Daniel Jonas , Schönberger Alvarez Ariel A. , te Heesen Pia , Lehrheuer Bastian , Pischinger Stefan , Hollert Henner , Roß-Nickoll Martina , Du Miaomiao
Published: March 2024

Gaseous and semi-volatile organic compounds emitted by the transport sector contribute to air pollution and have adverse effects on human health. To reduce harmful effects to the environment as well as to humans, renewable and sustainable bio-hybrid fuels are explored and investigated in the cluster of excellence “The Fuel Science Center” at RWTH Aachen University. However, data on the effects of bio-hybrid fuels on human health is scarce, leaving a data gap regarding their hazard potential. To help close this data gap, this study investigates potential toxic effects of a Ketone-Ester-Alcohol-Alkane (KEAA) fuel blend on A549 human lung cells. Experiments were performed using a commercially available air-liquid interface exposure system which was optimized beforehand. Then, cells were exposed at the air-liquid interface to 50-2000 ppm C3.7of gaseous KEAA for 1 h. After a 24 h recovery period in the incubator metabolic activity and cytotoxicity of cells were assessed. Our data support the international occupational exposure limits of the single KEAA constituents and moreover indicate no adverse effect to A549 cells when exposed to a fuel mixture. This finding applies only to the exposure scenario tested in this study and is difficult to extrapolate to the complexin vivosituation.
Changes in plant physiology during cultivation periods influence the hydrogen isotope ratio of the water-insect relationship
Authors: Fujii Tomohisa , Akiduki Gaku , Yabusaki Shiho , Tayasu Ichiro
Published: March 2024

The hydrogen stable isotope ratios (δ2H) in tissues of terrestrial insects are widely applied to estimate natal origins in field populations. The hydrogen isotopes of insect tissues incorporate those of environmental waters through the insects’ metabolic processes. Water sources and abiotic environmental factors reflect changes in plant physiology, as indicated by the δ2H values of plants. However, the influence of plant physiology on the assimilation of hydrogen in insect tissues derived from water through feeding diets is still unknown.

We experimentally examined the influence of water on the δ2H values of maize leaf and of forewings ofMythimna separata(Lepidoptera: Noctuidae) andSpodoptera frugiperda(Noctuidae). We prepared five specific water samples for cultivating maize, aiming to replicate the gradient of δ2H values observed in environmental waters across the Japanese archipelago.

The mean-percentage contribution of water to hydrogen in maize leaves was 17.6% (July-August) and 25.1% (September-October). Linear analyses indicated that 17.4% and 32.7% of hydrogen inM. separataandS. frugiperdaforewings were derived from water through the consumption of maize leaves. The slope values of linear regression between the insect forewings and the maize leaves supplied in the final instar were closest to 1.0. These results indicated that the δ2H values of maize leaves and insect forewings reflected those of water resources.

This study presented the seasonal changes in climate conditions that affected the δ2H values of host plants and insect tissues. Changes in plant physiology with seasonal variations may influence the interpretation of the linear relationship between water and insect tissues on the estimation of natal origins.
Integrative Co-Registration of Elemental Imaging and Histopathology for Enhanced Spatial Multimodal Analysis of Tissue Sections through TRACE
Authors: Lu Yunrui , Steiner Ramsey , Han Serin , Srivastava Aruesha , Shaik Neha , Chan Matthew , Diallo Alos , Punshon Tracy , Jackson Brian , Kolling Fred , Vahdat Linda , Vaickus Louis , Marotti Jonathan , Ho Sunita , Levy Joshua
Published: March 2024

Elemental imaging provides detailed profiling of metal bioaccumulation, offering more precision than bulk analysis by targeting specific tissue areas. However, accurately identifying comparable tissue regions from elemental maps is challenging, requiring the integration of hematoxylin and eosin (H&E) slides for effective comparison. Facilitating the streamlined co-registration of Whole Slide Images (WSI) and elemental maps, TRACE enhances the analysis of tissue regions and elemental abundance in various pathological conditions. Through an interactive containerized web application, TRACE features real-time annotation editing, advanced statistical tools, and data export, supporting comprehensive spatial analysis. Notably, it allows for comparison of elemental abundances across annotated tissue structures and enables integration with other spatial data types through WSI co-registration.

Availability and Implementation

Available on the following platforms– GitHub:jlevy44/trace_app, PyPI:trace_app, Docker:joshualevy44/trace_app, Singularity:joshualevy44/trace_app.

Contact

joshua.levy@cshs.org

Supplementary information

Supplementary data are available.
Inference of Transcriptional Regulation From STARR-seq Data
Authors: Safaeesirat Amin , Taeb Hoda , Tekoglu Emirhan , Morova Tunc , Lack Nathan A. , Emberly Eldon
Published: March 2024

One of the primary regulatory processes in cells is transcription, during which RNA polymerase II (Pol-II) transcribes DNA into RNA. The binding of Pol-II to its site is regulated through interactions with transcription factors (TFs) that bind to DNA at enhancer cis-regulatory elements. Measuring the enhancer activity of large libraries of distinct DNA sequences is now possible using Massively Parallel Reporter Assays (MPRAs), and computational methods have been developed to identify the dominant statistical patterns of TF binding within these large datasets. Such methods are global in their approach and may overlook important regulatory sites which function only within the local context. Here we introduce a method for inferring functional regulatory sites (their number, location and width) within an enhancer sequence based on measurements of its transcriptional activity from an MPRA method such as STARR-seq. The model is based on a mean-field thermodynamic description of Pol-II binding that includes interactions with bound TFs. Our method applied to simulated STARR-seq data for a variety of enhancer architectures shows how data quality impacts the inference and also how it can find local regulatory sites that may be missed in a global approach. We also apply the method to recently measured STARR-seq data on androgen receptor (AR) bound sequences, a TF that plays an important role in the regulation of prostate cancer. The method identifies key regulatory sites within these sequences which are found to overlap with binding sites of known co-regulators of AR.

1

Author Summary

We present an inference method for identifying regulatory sites within a putative DNA enhancer sequence, given only the measured transcriptional output of a set of overlapping sequences using an assay like STARR-seq. It is based on a mean-field thermodynamic model that calculates the binding probability of Pol-II to its promoter and includes interactions with sites in the DNA sequence of interest. By maximizing the likelihood of the data given the model, we can infer the number of regulatory sites, their locations, and their widths. Since it is a local model, it can in principle find regulatory sites that are important within a local context that may get missed in a global fit. We test our method on simulated data of simple enhancer architectures and show that it is able to find only the functional sites. We also apply our method to experimental STARR-seq data from 36 androgen receptor bound DNA sequences from a prostate cancer cell line. The inferred regulatory sites overlap known important regulatory motifs and their ChIP-seq data in these regions. Our method shows potential at identifying locally important functional regulatory sites within an enhancer given only its measured transcriptional output.
Masitinib limits neuronal damage, as measured by serum neurofilament light chain concentration, in a model of neuroimmune-driven neurodegenerative disease
Authors: Hermine Olivier , Gros Laurent , Tran Truong-An , Loussaief Lamya , Flosseau Kathleen , Moussy Alain , Mansfield Colin D. , Vermersch Patrick
Published: March 2024

Background

Masitinib is an orally administered tyrosine kinase inhibitor that targets activated cells of the innate neuroimmune system. We have studied the neuroprotective action of masitinib on the manifestations of experimental autoimmune encephalitis (EAE) induced axonal and neuronal damage. EAE is a model of neuroimmune-driven chronic neuroinflammation and therefore highly relevant to masitinib’s mechanism of action in neurodegenerative diseases. Importantly, neuronal damage, or prevention thereof, can be rapidly assessed by measuring serum neurofilament light chain (NfL) concentration in EAE-induced mice.

Methods

EAE induction was performed in healthy female C57BL/6 mice via active MOG35-55peptide immunization. Treatments were initiated 14 days post EAE induction. On day-0, 39 mice with established EAE symptoms were randomly assigned to 3 treatment groups (n=13): EAE control, masitinib 50 mg/kg/day (M50), and masitinib 100 mg/kg/day (M100). Treatment started on day-1 and ended on day-15. Blood samples were collected at day-1, day-8 (via tail vein sampling) and day-15 (via intracardiac puncture). Assessments included quantification of serum NfL levels along the disease duration, cytokine quantification at day-15, and clinical assessments.

Results

Masitinib treatment significantly (p<0.0001) limited NfL production with respect to control; specifically, relative change in serum NfL concentration at day-8 was 43% and 60% lower for the M50 and M100 groups, respectively. Likewise, for the assessment of absolute serum NfL at day-8 and day-15, there was a significantly lower NfL concentration for masitinib treatment as compared with control. Furthermore, EAE mice treated with masitinib showed significantly lower concentrations of several well-established pro-inflammatory cytokines relative to control at day-15. A beneficial effect of masitinib on functional performance was also observed, with both M50 and M100 groups showing significantly less relative deterioration in grip strength at day-15 as compared with control (p<0.001).

Conclusion

This study is the first demonstration that masitinib, a drug that targets the innate as opposed to the adaptive neuroimmune system, can lower serum NfL levels, and by extension therefore, neuronal damage, in a neuroimmune-driven neurodegenerative disease model. Overall, findings indicate that masitinib has a neuroprotective effect under conditions of chronic neuroinflammation and therefore plausible disease-modifying activity across a broad range of neurodegenerative diseases.
Bridging fungal resistance and plant growth through constitutive overexpression ofThchit42gene inPelargonium graveolens
Authors: Khatoon Kahkashan , Warsi Zafar Iqbal , Singh Akanksha , Singh Kajal , Khan Feroz , Singh Palak , Shukla Rakesh Kumar , Verma Ram Swaroop , Singh Munmun K. , Verma Sanjeet K. , Husain Zakir , Parween Gazala , Singh Pooja , Afroz Shama , Rahman Laiq Ur
Published: March 2024

Pelargonium graveolensessential oil possesses significant attributes, known for perfumery and aromatherapy. However, optimal yield and propagation are predominantly hindered by biotic stress. All biotechnological approaches have yet to prove effective in addressing fungal resistance. The current study developed transgenic geranium bridging molecular mechanism of fungal resistance and plant growth by introducing cassette 35S::Thchit42. Furthermore, 120 independently putative transformed explants were regenerated on kanamycin fortified medium. Primarily transgenic lines were demonstrated peak pathogenicity and antifungal activity against formidableColletotrichum gloeosporioidesandFusarium oxysporum. Additionally, phenotypic analysis revealed ∼2fold increase in leaf size and ∼2.1fold enhanced oil content. To elucidate the molecular mechanisms for genotypic cause,De novotranscriptional profiles were analyzed to indicate that the auxin-regulated longifolia gene is accountable for augmentation in leaf size, and ZF RICESLEEPER attributes growth upregulation. Collectively, data provides valuable insights into unravelling the mechanism ofThchit42-mediated crosstalk between morphological and chemical alteration in transgenic plants. This knowledge might create novel opportunities to cultivate fungal-resistant geranium throughout all seasons to fulfil demand.
Beyond functional connectivity: deep learning applied to resting-state fMRI time series in the prediction of 58 human traits in the HCP
Authors: Vieira Bruno Hebling , Schöttner Mikkel , Calhoun Vince D , Salmon Carlos Ernesto Garrido
Published: March 2024

Machine learning has made several inroads into the study of brain-behavior relations based on in vivo imaging. While the advent of deep neural networks was expected to further improve predictions, the current literature based on resting-state functional connectivity presents mixed results. We hypothesize that the representation of the data,i.e.in the form of functional connectivity, could restrict an advantage of deep learning techniques, namely that of learning complex representations directly from the data. Thus, we investigated if bypassing this feature extraction resulted in improved performance in the prediction of 58 widely studied behavioral traits from a large sample of Human Connectome Project subjects, using deep learning techniques. For this task, we adapted the InceptionTime architecture, which jointly predicts traits directly from regional time series through representation learning, and compared results with a strong kernel-based baseline. Results revealed that both models achieve comparable performance in most traits. Eleven significant differences in mean squared error were detected, however, with seven favoring the neural network approach, and this number increased when accounting for covariates. We additionally show that contrary to the expectation, the neural network approach was more robust to reductions in the training set size. On the other hand, it was more sensitive to reductions in the length of the time series at test time. Our results present a more nuanced view of the potential of deep learning for the prediction of behavior from neuroimaging, which allows learning features directly from the data.
Two opposing roles for Bmp signalling in the development of electrosensory lateral line organs
Authors: Campbell Alexander S. , Minařík Martin , Franěk Roman , Vazačová Michaela , Havelka Miloš , Gela David , Pšenička Martin , Baker Clare V. H.
Published: March 2024

The lateral line system enables all fishes and aquatic-stage amphibians to detect local water movement via mechanosensory hair cells in neuromasts, and many species to detect weak electric fields via electroreceptors (modified hair cells) in ampullary organs. Both neuromasts and ampullary organs develop from lateral line placodes. However, the molecular mechanisms underpinning ampullary organ formation are understudied relative to neuromasts, as the ancestral lineages of zebrafish (teleosts) andXenopus(frogs) independently lost electroreception. We identifiedBmp5as a promising candidate via differential RNA-seq in an electroreceptive ray-finned fish, the Mississippi paddlefish (Polyodon spathula; Modrell et al., 2017,eLife6: e24197). In an experimentally tractable relative, the sterlet sturgeon (Acipenser ruthenus), we found thatBmp5and four other Bmp pathway genes are expressed in the developing lateral line, and that Bmp signalling is active. Furthermore, CRISPR/Cas9-mediated mutagenesis targetingBmp5in G0-injected sterlet embryos resulted in fewer ampullary organs. Conversely, when Bmp signalling was inhibited by DMH1 treatment shortly before the formation of ampullary organ primordia, supernumerary ampullary organs developed. These data suggest that Bmp5 promotes ampullary organ development, whereas Bmp signalling via another ligand(s) prevents their overproduction. Taken together, this demonstrates two opposing roles for Bmp signalling during ampullary organ formation.
A perivascular niche supports endometrial epithelial regeneration
Authors: Li Shu-Yun , Whiteside Sarah , Li Bo , Sun Xiaofei , DeFalco Tony
Published: March 2024

Endometrial regeneration is essential for reproductive cycles and pregnancies, allowing the endometrium to undergo repair and renewal after menstruation and parturition. Epithelial cells lining the uterine cavity are shed during each cycle, although remnant luminal and glandular epithelial cells can regenerate the lumen lining. It is presumed that adult stem/progenitor cells in the uterine stroma also contribute to this regeneration. However, the specific cell type(s) and the underlying mechanisms have not been determined. Herein, we use genetic lineage tracing assays in mice to identifyNestin+ perivascular cells as active contributors to epithelial regeneration. Notch signaling maintainsNestin+ perivascular cells in a quiescent state but these cells re-enter the cell cycle and differentiate into epithelial cells via estrogen-stimulated suppression of Notch signaling dependent on estrogen receptor alpha (ERα/ESR1). These findings demonstrate that perivascular cells support epithelial regeneration and reveal a mechanism regulating the quiescence and activation of uterine perivascular cells.
Scalable Isolation of Surface-Engineered Extracellular Vesicles and Separation of Free Proteins via Tangential Flow Filtration and Size Exclusion Chromatography (TFF-SEC)
Authors: Kawai-Harada Yuki , Nimmagadda Vasudha , Harada Masako
Published: March 2024

Background

Extracellular vesicles (EVs) represent small lipid bilayer structures pivotal in mediating intercellular communication via biomolecular transfer. Their inherent characteristics, including packaging, non-immunogenicity, and biofluid stability, position EVs as promising drug delivery vectors. However, developing clinical quality EVs requires multifaceted technological advancement.

Methods

In this study, a method is introduced for engineering extracellular vesicles (eEVs) from cultured cells and their subsequent isolation using lab-scale tangential flow filtration (TFF). This is the first study to evaluate DNA loading efficacy into EVs isolated by TFF, marking a significant milestone in the field of targeted drug delivery. Initially, cells are transfected with EV-display constructs to facilitate the secretion of eEVs bearing the desired coding molecules. Following brief centrifugation, the cell culture media undergoes filtration using hollow fiber filters. TFF, by applying a constant flow, effectively segregates molecules based on designated molecular weight cut-off (MWCO), enriching particles between 50 nm and 650 nm.

Results

Compared to conventional methods like ultracentrifugation, TFF demonstrates higher efficiency in removing undesired molecules/aggregates while exerting less stress on EVs. Characterization of eEVs through various assays confirms TFF’s superiority in isolating pure EV populations. Additionally, the necessity of size-exclusion chromatography (SEC) after tangential flow filtration (TFF) becomes evident for effectively removing unbound protein contaminants.

Conclusion

In conclusion, TFF-SEC emerges as a scalable and superior approach for eEV isolation, promising significant advancements in clinical applications.
Cool ocean temperatures fail to buffer the impacts of heat exposure during low tide on the behaviour and physiology of a keystone predator
Authors: Walton Lydia N. , Watts Viola R. , Schuster Jasmin M. , Bates Amanda E.
Published: March 2024

Air temperatures are warming at faster rates than ocean temperatures, and this “land-sea warming contrast” may create reprieves from thermal stress by providing cool underwater refugia during extreme heat events. Here we tested the impacts of the “land-sea warming contrast” on physiology (metabolism) and behaviour (feeding) in the juvenile life stage of a keystone intertidal predator,Pisaster ochraceus, by experimentally manipulating air (∼20℃, 25℃, 30℃) and water (∼15℃, 20℃) temperatures (at independent rates) representing early summer, late summer, and heatwave conditions in Barkley Sound (British Columbia, Canada). We further made observations of air temperatures, sea surface temperatures, andPisastermoribundity at our study location to support interpretation of our results. We predicted metabolism and feeding would increase with early and late summer temperatures, but decrease during heatwave conditions as animals surpass their thermal optimum. We observed the greatest mortality and lowest feeding in juvenilePisasterexposed to cool ocean temperatures (∼15℃) and high aerial temperatures typical of extreme heat events (∼30℃). Feeding rates increased with heat stress duration, indicating animals may be compensating for elevated metabolism. Metabolic rates did not differ between air temperatures, but oxygen consumption was higher in animals with access to mussels than forPisasterthat were fasted. The highest levels of experimental and field moribundity were observed in August, indicatingPisastermay have accumulated physiological stress damage following elevated air and ocean temperatures throughout the summer. Our research implicates shifts in community dynamics due to the loss of this keystone species as air temperatures warm.

Summary Statement

Cooler ocean temperatures, rather than creating thermal refugia, may cause physiological stress for juvenilePisaster ochraceusexposed to warm air during low tide.
Labeling Natural Killer cells with superparamagnetic iron oxide nanoparticles for detection by preclinical and clinical-scale magnetic particle imaging
Authors: Sehl Olivia C. , Yang Yanwen , Anjier Ariana R , Nevozhay Dmitry , Cheng Donghang , Guo Kelvin , Fellows Benjamin , Mohtasebzadeh A. Rahman , Mason Erica E. , Sanders Toby , Kim Petrina , Trease David , Koul Dimpy , Goodwill Patrick W. , Sokolov Konstantin , Wintermark Max , Gordon Nancy , Greve Joan M. , Gopalakrishnan Vidya
Published: March 2024

Introduction

Clinical adoption of NK cell immunotherapy is underway for medulloblastoma and osteosarcoma, however there is currently little feedback on cell fate after administration. We propose magnetic particle imaging (MPI) for the detection, localization, and quantification of VivoTrax-labeled NK cells.

Methods

Human-derived NK-92 cells were labeled by co-incubation with VivoTrax for 24 hours then the excess nanoparticles were washed with centrifugation. Cytolytic activity of labeled vs. unlabeled NK-92 cells was assessed after 4 hours of co- incubation with medulloblastoma cells (DAOY) or osteosarcoma cells (LM7 or OS17) using bioluminescent or GFP counts. Labeled NK-92 cells at two different doses (0.5 or 1 x 106) were administered to excised mouse brains (cerebellum), tibias, and lungs then imaged by 3D preclinical MPI (MOMENTUM imager) and localized relative to fiducial markers. NK-92 cells were imaged by clinical-scale MPI under development at Magnetic Insight Inc.

Results

NK-92 cells were labeled with an average of 3.17 pg Fe/cell with no measured effects on cell viability or cytolytic activity against 3 tumor cell lines. MPI signal was directly quantitative with the number of VivoTrax-labeled NK-92 cells, with preclinical limit of detection of 3.1 x 104cells on MOMENTUM imager. Labeled NK-92 cells could be accurately localized in mouse brains, tibias, and lungs within < 1 mm of stereotactic injection coordinates with preclinical scanner. Feasibility for detection of a clinically relevant dose of 4 x 107labeled NK-92 cells was demonstrated on clinical-scale MPI.

Conclusion

MPI can provide sensitive, quantitative, and accurate spatial information on NK cell delivery, showing its potential to resolve a significant unmet clinical need to track NK cell treatments in patients.
Understanding the binding mechanisms of Alzheimer risk factor TREM2 in lipid bilayer using multi-scale molecular dynamics simulations
Authors: Zhong Zhiwen , Ulmschneider Martin , Lorenz Christian D.
Published: March 2024

Alzheimer’s disease (AD) is a widespread neurodegenerative condition affecting millions globally. Recent research has implicated variants of the triggering receptor ex-pressed in myeloid cells 2 (TREM2) as risk factors for AD. TREM2, an immunomodula-tory receptor on microglial surfaces, plays a pivotal role in regulating microglial activa-tion by associating with DNAX-activation protein 12 (DAP12). Despite its significance, the mechanism underlying the formation of the complex between the transmembrane domains (TMDs) of TREM2 and DAP12 remains unclear. This study employs multi-scale molecular dynamics (MD) simulations to investigate three TMD complex models, including two derived from experiments and one generated by AlphaFold2. Conducted within a lipid membrane consisting of an 80:20 mixture of phosphatidylcholine (POPC) and cholesterol, our analysis reveals hydrogen bonding interactions between K26 of TREM2 and D16 of DAP12 in all three models, consistent with previous experimen-tal findings. Our results elucidate the different spatial conformations observed in the models and offer insights into the structure of the TREM2/DAP12 TMD complex. Furthermore, we elucidate the role of charged residues in the assembly structure of the complex within the lipid membrane. These findings enhance our understanding of the molecular mechanism governing TREM2/DAP12 complex formation, providing a foundation for designing novel therapeutic strategies to address AD and other neu-rodegenerative diseases.

Graphical Abstract

Highlights

Different TREM2/DAP12 complexes are generated using experimental PDB structures or AlphaFold2

Long time coarse-grained MD simulations are used for getting the sys-tems stable

Atomistic detailed complex structures are captured using all-atom sim-ulation

Provides mechanistic insight into TREM2 transformation from its un-bound state to bounded state

Offers novel insights into the molecular basis of TREM2/DAP12 sig-nalling pathway
Effect of Electroacupuncture Pretreatment on Cognitive Impairment and Mitophagy in Aging Model Rats and its mechanism
Authors: Liu Biyong , Tan Tiantian , Liu Jianmin , Li Zhijie , Feng Qunhu , Qiu Su , Xiong Chengkai , Liu Qing , Li Jialin , Li Yihong
Published: March 2024

Objective

Investigate the effects of "Shuanggu Yitong" EA pretreatment on cognitive impairment, mitochondrial function, and mitophagy in aging model rats, and to analyze the related mechanisms.

Methods

Forty 3-month-old male SD rats were randomly divided into a blank group, a model group, an EA group, and a sham EA group, with 10 rats in each group. And the Morris water maze test was performed after the intervention. HE staining, to observe the morphological changes of the hippocampus of the model rats. Nissl staining was used to observe the changes in the number of hippocampal neurons in rats, Western Blotting (WB) was used to observe the expression of endogenous PTEN-induced hypothetical kinase 1 (PINK1) and human Parkinson’s protein 2 (Parkin) in the hippocampus, spectrophotometry was employed to detect the activity of respiratory chain complex I in the hippocampus of the model rats, and flow cytometry was utilized to detect hippocampal mitochondrial membrane potential (MMP) and hippocampal mitochondrial permeability transition pore (MPTP) opening.

Results

"Shuanggu Yitong" EA pretreatment relieved the cognitive impairment induced by D-galactose in subacute aging model rats. The mechanism of "Shuanggu Yitong" EA pretreatment in the improvement of cognitive impairment of subacute aging model rats may be related to the enhancement of Pink1/Parkin mediated mitophagy and the timely removal of accumulated abnormal mitochondria, thus improving mitochondrial function.

Conclusion

"Shuanggu Yitong" EA pretreatment can significantly improve the cognitive impairment induced by D-galactose in subacute aging model rats.
Alcohol, smoking, and illicit substance use in Cameroon: unveiling related risk factors among university students in Yaoundé
Authors: Lekeumo Cheuyem Fabrice Zobel , Edzamba Michel Franck , Amani Adidja , Mossus Tatiana
Published: March 2024

Background

Substance use, including alcohol, tobacco and illicit drugs, is a growing public health problem worldwide. There is a rapid increase in substance use among young adults in many sub-Saharan African countries. This study aimed to assess the factors associated with the use of different psychoactive substances among university students in Yaoundé.

Methods

A cross-sectional and analytical study was conducted from September to October 2023 at Yaoundé 1 University in Cameroon. All eligible students aged 18 years and older who gave written informed consent were included. A convenience nonprobability sampling method was used to recruit consenting students. The data collectors were medical students who were trained for 2 days and given appropriate instructions before the survey. The data collected were reviewed and checked for completeness before being entered. The data were analyzed using Statistics 4.3.1.

Results

A total of 191 university students were enrolled in the study. Age (p-value=0.002), level of study (p-value=0.048), and smoking status (p-value=0.005) of the participants were significant factors associated with alcohol on univariate analysis. Multivariate logistic regression showed that students aged 20-25 years were significantly 2.9 times more likely to drink alcohol than those aged less than 20 years (p-value=0.003). Students who smoke were 2.7 times more likely to drink alcohol than those who do not smoke (p-value=0.008). Living situation (p=0.013) and drug use status (p-value<0.0001) were significant factors associated with smoking on univariate analysis. On multivariate analysis, drug users were 3.2 times more likely to smoke than drug non-users (p-value<0.0001). Drug use was significantly associated with district of residence of consumer on univariate analysis (p-value=0.024). Living situation (p-value=0.016), faculty/school(p-value=0.04), and district of residence (p-value=0.037) were significantly associated with polysubstance use. Students living in shared accommodation were 3.8 times more likely to be polysubstance users than those living with their families (p-value=0.023). Almost all smokers (95.1%) reported being aware the of the psychosocial, mental and health consequences of substance use (p-value=0.021).

Conclusion

Several factors have been associated with substance use among college students. These sociodemographic factors can help to strategize and implement tailored interventions to reduce the risk of subsequent substance dependence and other harmful consequences.
Human primosome requires replication protein A when copying DNA with inverted repeats
Authors: Baranovskiy Andrey G. , Morstadt Lucia M. , Romero Eduardo E. , Babayeva Nigar D. , Tahirov Tahir H.
Published: March 2024

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA polymerases delta and epsilon (Polε). Replication protein A (RPA) tightly binds to single-stranded DNA strands, protecting them from nucleolytic digestion and unauthorized transactions. We report here that RPA plays a critical role for the human primosome during DNA synthesis across inverted repeats prone to hairpin formation. On other alternatively structured DNA, forming a G-quadruplex, RPA does not assist primosome. A stimulatory effect of RPA on DNA synthesis across hairpins was also observed for the catalytic domain of Polα but not of Polε. The winged helix-turn-helix domain of RPA is essential for an efficient hairpin bypass and increases RPA-Polα cooperativity on the primed DNA template. Cryo-EM studies revealed that this domain is mainly responsible for the interaction between RPA and Polα. The flexible mode of RPA-Polα interaction during DNA synthesis implies the mechanism of template handover between them when the hairpin formation should be avoided. This work provides insight into a cooperative action of RPA and primosome on DNA, which is critical for DNA synthesis across inverted repeats.

SIGNIFICANCE

This work revealed the critical role of RPA during DNA synthesis across inverted repeats by the human primosome. It was shown that a small winged helix-turn-helix domain of RPA is essential for the cooperative action of RPA and primosome, especially when copying DNA sequences prone to hairpin structure formation. Structural studies uncovered the mode of RPA and Polα integration, which assures an efficient DNA handover between them. This work provides a notable insight into the early steps of Okazaki fragments synthesis.
Structural basis of substrate transport and drug recognition by the human thiamine transporter SLC19A3
Authors: Gabriel Florian , Spriestersbach Lea , Fuhrmann Antonia , Jungnickel Katharina E. J. , Mostafavi Siavash , Pardon Els , Steyaert Jan , Löw Christian
Published: March 2024

Thiamine (vitamin B1) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitaminde novoand thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane which is mainly mediated by two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters leads to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report eight cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enabled us to capture different states of SLC19A3’s transport cycle, revealing the molecular details of thiamine recognition and transport. We identified nine novel drug interactions of SLC19A3 and determined structures of the transporter in complex with the inhibitors fedratinib, hydroxychloroquine, amprolium and amitriptyline. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.
Targeted protein degradation of the CPSF complex by benzoxaboroles through sumoylation
Authors: Zhang Ning , Samad Abdul , Zahedifard Farnaz , Wall Richard J. , Horn David , Zoltner Martin , Field Mark C.
Published: March 2024

Benzoxaboroles (BoBs) feature a boron-heterocyclic core and are an important innovation in the development of drugs against a range of pathogens and other pathologies. A broad spectrum of pharmacology is associated with chemically diverse BoB derivatives and includes multiple modes-of-action (MoA) and targets. However, a consensus MoA for BoBs targeting evolutionarily diverse protozoan pathogens has emerged with the identification of CPSF3/CPSF73 in the CPSF complex in both apicomplexan and kinetoplastida parasites. Here we establish a functional connection between protein sumoylation and the boron-heterocyclic scaffold shared by all BoBs using comprehensive genetic screens inTrypanosoma brucei.There is a rapid temporal and spatial shift in global protein sumoylation following BoB exposure and members of the CPSF complex are specifically destabilised in a SUMO and proteosome-dependent manner. Finally, we find rapid decrease in bulk mRNA levels, consistent with the role of CPSF3 in mRNA maturation. We propose that a combination of direct inhibition coupled with targeted degradation of CPSF3 underpins the specificity of BoBs against trypanosomatids.
Combinatorial control of type IVa pili formation by the four polarized regulators MglA, SgmX, FrzS and SopA
Authors: Oklitschek Michel , Carreira Luís António Menezes , Muratoğlu Memduha , Søgaard-Andersen Lotte , Treuner-Lange Anke
Published: March 2024

Type IVa pili (T4aP) are widespread and enable bacteria to translocate across surfaces. T4aP engage in cycles of extension, surface adhesion and retraction, thereby pulling cells forward. Accordingly, the number and localization of T4aP are critical to efficient translocation. Here, we address how T4aP formation is regulated inMyxococcus xanthus, which translocates with a well-defined leading and lagging cell pole using T4aP at the leading pole. This localization is orchestrated by the small GTPase MglA and its downstream effector SgmX that both localize at the leading pole and recruit the PilB extension ATPase to the T4aP machinery at this pole. Here, we identify the previously uncharacterized protein SopA and show that it interacts directly with SgmX, localizes at the leading pole, stimulates polar localization of PilB, and is important for T4aP formation. We corroborate that MglA also recruits FrzS to the leading pole, and that FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP formation and T4aP-dependent motility in various mutants support a model whereby the main pathway for stimulating T4aP formation is the MglA/SgmX pathway. FrzS stimulates this pathway by recruiting SgmX and SopA. SopA stimulates the MglA/SgmX pathway by stimulating the function of SgmX, likely by promoting the SgmX-dependent recruitment of PilB. The architecture of the MglA/SgmX/FrzS/SopA protein interaction network for orchestrating T4aP formation allows for combinatorial regulation of T4aP levels at the leading cell pole resulting in discrete levels of T4aP-dependent motility.