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Huntington's disease (HD) is a fatal neurodegenerative disease characterized by the expression of huntingtin protein (htt) that has a polyglutamine (CAG; polyQ) repeat domain consisting of 36 or more glutamines (mhtt). Historically, mhtt is more broadly associated with HD severity, as are elevated metal levels observed in HD patients. The depletion of wild-type (WT) htt (fewer than 36Qs) is also recognized as a contributing factor to HD progression; however, many questions remain about the interactions of biorelevant metals with WT htt and the impact of the interactions on protein aggregation. In the present work, we utilize a combination of biochemical assays and spectroscopic techniques to provide insights into the interaction of copper with an in vitro htt model (N171-17Q). Herein, we use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering to show that the addition of equimolar or higher concentrations of Cu(II) to htt induces time- and temperature-dependent protein oligomerization/aggregation. Additionally, chelation assays, trapped ion mobility spectrometry, and mass spectrometry confirm the (i) rapid reduction of Cu(II) in the presence of N171-17Q htt, (ii) direct binding of multiple copper ions per protein, and (iii) complex Cu:htt speciation profile with a preference for three distinct Cu:htt states. These findings contribute to our molecular level understanding of copper's role in the depletion and oligomerization/aggregation of WT htt while underscoring the physiological significance of our work, its potential relevance to metal binding in mhtt, and its significance for identifying new avenues for biomarker exploration and therapeutic design strategies.

Cabotegravir is a novel therapeutic option for HIV prevention. Similar to the opioid morphine, cabotegravir, undergoes glucuronidation through the enzymes uridine diphosphate glucuronosyltransferase (UGT) in the liver. We hypothesize that their combination could lead to drug-drug interactions, and this notion was explored in both male and female mice. Our findings indicate a better analgesic response to morphine in females compared to male animals, which was to be mediated by μ-opioid receptors and proteins associated with synaptic plasticity. Co-administration with cabotegravir appears to intensify morphine concentrations in the brain and the analgesic response in male animals only. Moreover, cabotegravir-induced fluctuations in the expression of the UGT enzymes correlated with alterations in drug metabolism and excretion and in the production of inflammatory cytokines primarily driven by morphine in the brains and cabotegravir in the liver. The increased levels of inflammatory cytokines in males aligned with noticeable morphological changes in the liver. In summary, co-exposure with cabotegravir changed the biodistribution in the brain, affected liver metabolism, and altered kidney excretion, leading to changes in gene expression and inflammatory effects that could disrupt morphine analgesia responses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nazira El-Hage, PhD reports financial support was provided by National Institute of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Masson SAS.)

The goal of this study was to calculate the cost of purulent vaginal discharge (PVD) in dairy cows. The dataset included 11,051 cows from 16 dairy herds located in 4 regions of the United States. Purulent vaginal discharge was characterized as a mucopurulent, purulent, or reddish-brownish vaginal discharge collected at 28 ± 7 DIM. Gross profit was calculated as the difference between incomes and expenses, and the cost of PVD was calculated by subtracting the gross profit of cows with PVD from the gross profit of cows without PVD. Continuous outcomes such as milk production (kg/cow), milk sales ($/cow), cow sales ($/cow), feed costs ($/cow), reproductive management costs ($/cow), replacement costs ($/cow), and gross profit ($/cow) were analyzed using linear mixed effects models. Pregnancy and culling by 305 DIM were analyzed by generalized linear mixed effects models using logistic regression. Models included the fixed effects of PVD, metritis, parity, region, season of calving, and morbidity in the first 60 DIM, as well as the interactions between PVD and metritis, PVD and parity group, and PVD and morbidity. Farm and the interaction between PVD and farm were considered random effects in all the statistical models. A stochastic analysis was conducted using 10,000 iterations with varying relevant inputs. Cows with PVD produced less milk (9,753.2 ± 333.6 vs. 9,994.6 ± 330.9 kg/cow), were less likely to be pregnant (70.7 ± 1.7% vs. 78.9 ± 1.2%), and were more likely to be culled by 305 DIM (34.6 ± 1.7% vs. 27.2 ± 1.3%) compared with cows without PVD. Consequently, milk sales (4,744.7 ± 162.3 vs. 4,862.1 ± 161.0 $/cow) and residual cow value (1,079.6 ± 23.0 vs. 1,179.3 ± 20.3 $/cow) were lesser for cows with PVD. Replacement (639.4 ± 26.4 vs. 526.0 ± 23.4 $/cow) and reproductive management costs (76.3 ± 2.5 vs. 69.0 ± 2.4 $/cow) were greater for cows with PVD. The mean cost of PVD was $202. The stochastic analysis also showed a mean cost of $202, ranging from $152 to $265. The robust dataset and the stochastic analysis strengthen both the external and internal validity of our findings, offering a deeper understanding of the economic consequences of PVD. In conclusion, PVD resulted in large economic losses to dairy herds by being associated with decreased milk yield, impaired reproduction, and greater culling., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

This study evaluated the prediction that tDCS-augmented in vivo exposure (IVE) for specific fears would result in durable changes in heart rate (BPM) and heart-rate variability (HRV) during and just after exposure to feared targets. In a double-blind, placebo-controlled trial, participants with contamination- and animal phobia (N = 49) were randomized to active tDCS (1.7 mA, 20 min; n = 27), or sham tDCS (1.7 mA, 30 s; n = 22), followed by a single session of 30 min of IVE. Active tDCS targeted excitation of the left mPFC and inhibition of the right dlPFC. BPM and HRV were acquired during behavioral approach tasks involving brief (30 s) exposure to feared targets at pre-treatment, post-treatment, and a 1-month follow-up, as well as during six 5-min. trials of exposure. Active tDCS produced significantly greater reductions in BPM, and marginally greater increases in HRV from pre-treatment to 1-month in an extinction context, compared to sham tDCS. Similarly, active tDCS produced significantly greater reductions in BPM, and increases in HRV during IVE, relative to the sham tDCS group. Findings for the generalization context were non-significant. Consistent with the main outcome findings, tDCS may offer an effective means of enhancing outcomes in exposure therapy, perhaps through top-down modulation of autonomic arousal., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Leprosy is a dermatoneurological disease and can cause irreversible nerve damage. In addition to being able to mimic different rheumatological, neurological and dermatological diseases, leprosy is underdiagnosed because several professionals present lack of training. The World Health Organization instituted active search for new leprosy cases as one of the four pillars of the zero-leprosy strategy. The Leprosy Suspicion Questionnaire (LSQ) was created aiming to be a screening tool to actively detect new cases; it is composed of 14 simple yes/no questions that can be answered with the help of a health professional or by the very patient themselves. During its development, it was noticed that the combination of marked questions was related to new case detections. To better encapsulate and being able to expand its use, we developed MaLeSQs, a Machine Learning tool whose output may be LSQ Positive when the subject is indicated for being further clinically evaluated or LSQ Negative when the subject does not present any evidence that justify being further evaluated for leprosy. To achieve a reasonable product, we trained four classifiers with different learning paradigms, Support Vectors Machine, Logistic Regression, Random Forest and XGBoost. We compared them based on sensitivity, specificity, positive predicted value, negative predicted value, and area under the ROC curve. After the training process, the Support Vectors Machine was the classifier with the most balanced metrics of 85.7% sensitivity, 69.2% specificity, 18.6% precision, 98.3% negative predicted values and an area under the ROC curve of 0.775, and it was chosen as the MaLeSQs. With Shapley values, we were able to evaluate variable importance and nerve symptoms were considered important to differentiate between subjects that potentially had leprosy from those who did not., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Dairy farmers are interested in reducing the carbon footprint of milk. Reducing methane (CH 4 ) emissions is a key part of this goal, and manure is a significant CH 4 source. Technologies like anaerobic digesters for biogas production are effective; however, adoption rates are slowed by upfront costs and infrastructure needs. Achieving near-term emission reductions needs low-cost alternatives that can be quickly and widely adopted. Previous studies have shown that "acidification" of manure by adding sulfuric acid (H 2 SO 4 ) suppressed CH 4 emissions; however, widespread adoption may be hindered by the challenge of handling acid on farms. This laboratory study was performed for 157 days at 24°C, and compared the efficacy of a sulfate-based non-acidic fertilizer (CaSO 4 ), and two rates of acidification, one at pH > 7 and one at pH < 7, for a sulfate-based acid (H 2 SO 4 ) and a sulfate-free acid (H 3 PO 4 ). Methane suppression by CaSO 4 at multiple rates was also analyzed. Two mechanisms of suppression were observed: acidification had a demonstrable early effect, lowering cumulative CH 4 emission within 40 days by up to 65% for H 2 SO 4 and 54% for H 3 PO 4 , while sulfate-containing compounds showed increasing suppression after 50 days. Final cumulative CH 4 suppression was up to 63% for CaSO 4 and 91% for H 2 SO 4 , while H 3 PO 4 was least effective. These results suggest H 2 SO 4 is highly effective due to the combination of acidity and sulfate. Adding sulfate alone (CaSO 4 ) was more effective than adding acid alone (H 3 PO 4 ). Hence, sulfate-based additives-like gypsum-may hold promise as an alternative near-term solution for dairy farms to make large CH 4 reductions., (© 2025 His Majesty the King in Right of Canada and The Author(s). Journal of Environmental Quality published by Wiley Periodicals LLC on behalf of American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. Reproduced with the permission of the Minister of Agriculture and Agri‐Food Canada.)

Genomic prediction of daughter pregnancy rate (GDPR) and cow conception rate (GCCR) are fertility traits developed to help improve selection for reproductive performance. Although these traits overlap, their denominator can vary, and programs with different strategies combining estrus detection (ED) and timed AI might experience different associations with these traits (e.g., shorter interval for first service for high GDPR quartiles, but not for GCCR). The objectives were to assess days from calving to first service (TP1), AI at ED (AIE), pregnancy at the first service (P1), pregnancy loss for the first service (PL), and number of services to conception (NSFC) in 2 reproductive programs (RepP) combining variable ED length and timed AI (TAI) and their relationship with GDPR and GCCR. Holstein cows from a single farm were randomly allocated to 2 different programs, the short RepP (n = 982) or long RepP (n = 942). In the short RepP, cows were enrolled in a Presynch-Ovsynch (PGF 2α : 36 ± 3 and 50 ± 3 DIM) followed by ED and AI from 50 ± 3 to 62 ± 3 DIM. Cows not detected in estrus underwent in the Ovsynch (GnRH: 62 ± 3, PGF 2α : 69 ± 3, GnRH: 71 ± 3 and TAI :72 ± 3 DIM). In the long RepP, cows received a PGF 2α at 50 ± 3 DIM followed by AI at ED up to 81 ± 3 DIM. Cows with no ED were enrolled in an Ovsynch (GnRH:82 ± 3, PGF 2α :89 ± 3, GnRH:91 ± 3 and TAI:92 ± 3 DIM). Cows were categorized into quartiles (Q1 to Q4) considering their GDPR (qGDPR) and GCCR (qGCCR). Statistical analyses included logistic regression used for AIE, P1, and PL; Poisson regression for the NSFC; and linear regression for TP1. Models included AIE, P1, PL, NSFC, and TP1 as dependent variables, with RepP and qGDPR for GDPR and RepP and qGCCR for GCCR as independent variables. Time to pregnancy was analyzed using Cox's proportional hazard model. The short RepP had a shorter TP1 (short = 64.3 vs. long = 72.1) and fewer NSFC than the long RepP (short = 2.9 vs. long = 3.1). The long RepP had a higher AIE (short = 45.2% vs. long = 73.2%). The P1 tended to be greater in the short than the long RepP (short = 33.7% vs. long = 30.0%). Cows in Q4 (GDPR and GCCR) had lower TP1 and NSFC, greater AIE and P1 compared with the lowest quartiles (Q1). Interactions between RepP and GDPR were present for TP1, AIE, and P1, but no interactions were observed between RepP and GCCR. The short program had a shorter interval from calving to pregnancy than the long program. Cows in the highest quartiles for GDPR and GCCR had shorter intervals from calving to pregnancy and higher pregnancy hazards than the lowest quartiles. The current study revealed that cows enrolled in long RepP that relied on longer ED intervals had lower reproductive outcomes than short RepP cows, and cows ranked in the highest GDPR and GCCR quartile for most responses had better outcomes independent of the RepP used., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

This study presents an analytical approach coupling novel ambient ionization sources with trapped ion mobility spectrometry (TIMS) and tandem mass spectrometry (MS/MS) for the rapid characterization of fentanyl analogs. Two ambient ionization sources were illustrated for minimal sample preparation and rapid analysis: electrospray ionization (nESI) and direct analysis in real time (DART). Fentanyl analogs can be separated using nESI-TIMS-MS/MS based on differences in their mobility and/or fragmentation pattern; reference mobility spectra are reported for 234 single standards. In contrast, DART-TIMS-MS/MS allowed for the characterization of 201 compounds due to differences in the protonation pattern and efficiency when compared to nESI. The TIMS high resolving power (R > 80) allowed baseline separation for most isomers and mobility trends were established for methylated and fluorinated isomers, with the more compact ortho-substituted analogs showing distinct separation from para- and meta-substituted species. This multi-dimensional strategy offers a comprehensive characterization of fentanyl analogs and other synthetic opioids with minimal sample preparation. This analysis shows significant potential for high-throughput screening (<5 min) and high sensitivity detection (

Background: Patients with chronic kidney disease (CKD) have serum, bone, and vascular abnormalities presenting as chronic kidney disease-mineral bone disorder (CKD-MBD) syndrome. This study sought to identify the parameters with the greatest relative impact on progression of CKD-MBD abnormalities., Materials and Methods: This prospective study measured 237 parameters including serum markers, clinical variables, dual-energy X-ray absorptiometry (DXA) measurements, vascular calcifications, and histomorphometric results from bone samples obtained at baseline and after 2 - 3 years. Relative impact of these parameters on kidney function, bone changes, and vascular calcification were assessed using machine learning, a subset of artificial intelligence analyses., Results: Baseline estimated glomerular filtration rate (eGFR) values ranged from 18 to 70 mL/min and declined in 52% of subjects by at least 3.3% annually during the study. These declines in eGFR were associated with changes in specific serum markers, bone quantity decreases, and bone quality alterations, but not with arterial calcifications. Arterial calcifications were associated with collagen crosslinking heterogeneity, serum phosphorus, diuretics and atorvastatin treatment, but not with kidney function. Baseline collagen crosslinking heterogeneity was an important factor impacting progression of coronary, but not aortic calcification. Baseline serum phosphorus was a factor primarily associated with progression of aortic calcification., Conclusion: Machine learning revealed specific bone and vascular abnormalities occurring early during loss of kidney function. Bone, vascular, blood, medication use, and other parameters were identified impacting the presence and progression of arterial calcification and altering bone quality and quantity in this understudied patient population. Serum phosphorus levels considered normal impacted progression of arterial calcification. Identification of these parameters and their relative importance enhances our understanding of CKD progression and should improve patient care.

Background: Converging evidence suggests that bipolar disorder (BD) involves mitochondrial dysfunction and prefrontal cortex (PFC) hypometabolism associated with cognitive impairment, which persists in remitted BD individuals. Transcranial infrared laser stimulation (TILS) provides safe, non-invasive brain stimulation that enhances PFC metabolism via photobiomodulation of mitochondrial respiration and tissue oxygenation. We tested the hypothesis that the neurocognitive deficits found in BD may be ameliorated by TILS treatments., Methods: This is the first study to explore neurocognitive effects of repeated TILS administration in BD. Using an open-label design, 29 individuals with remitted BD received six weekly TILS treatments. Working memory and attention were assessed with trail-making and 2-back tasks sensitive to TILS cognitive effects in individuals with BD. Changes in PFC network interactions were measured with functional near-infrared spectroscopy (fNIRS) because this method can measure TILS effects on oxygen metabolism in the PFC of individuals with BD., Results: Participants reported no adverse effects from treatment, confirming the safety of this intervention in individuals with BD. Cognitive test results showed that in people with remitted BD, TILS was effective at improving cognition, i.e., enhanced speed and accuracy in tasks reflecting cognitive flexibility, working memory, and attentional control. Antipsychotic medication improved TILS cognitive effects. The fNIRS results showed a significant reduction in PFC network correlations of oxygenated hemoglobin changes driven by cognitive task performance. The right-hemisphere frontopolar cortex showed greater TILS effects than its left-hemisphere counterpart., Conclusions: Repeated TILS is a safe intervention to improve cognition in people with remitted BD. Continued antipsychotic medication may have contributed to the cognitive improvement. To confirm TILS efficacy, a sham-controlled, double-blinded randomized trial is needed., Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05354895., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Barrett, Davis, Siegel-Ramsay, Bichlmeier, Almeida and Gonzalez-Lima.)

Feed efficiency is critical in dairy farming, impacting production costs and environmental sustainability. The development of the trait residual feed intake (RFI) has provided an opportunity to select dairy cows that are more efficient in converting nutrients into milk. Note that RFI requires individual daily intake records, which are typically collected on a limited number of research farms. In this context, the identification of biomarkers that can be used to identify and select more feed-efficient cows is of great interest. As such, this study aimed to identify ruminal and serum biomarkers associated with RFI in mid-lactation Holstein cows. A selected subset of 24 out of 454 Holstein cows was used in this study. This subset was strategically selected to represent extremes of least feed-efficient (LFE, n = 12, RFI = 2.44) and most feed-efficient (MFE, n = 12, RFI = -2.69) cows with no difference in the 3 energy sinks, namely body weight (BW) change, metabolic BW, and energy secreted in milk. Rumen fluid and serum samples were collected between 60 and 90 d in milk (DIM). Rumen fluid samples were collected using an oresophageal tubing procedure. Serum samples were used to measure fatty acids using a 2-step assay. The fatty acid methyl ester was assessed using solid phase extraction and quantified using chromatographic peak area and internal standard-based calculations. Ruminal ammonia nitrogen was measured using a phenol-hypochlorite assay, while serum urea was measured using a commercial ELISA assay. Cows in the MFE group had higher ruminal ammonia nitrogen concentrations than cows in the LFE group. There were no differences in serum urea concentration between MFE and LFE cows. Serum fatty acids concentration differed between groups, with myristic acid (C14:0), palmitic acid (C16:0), cis-heptadecenoic acid (cis9-17:1), stearic acid (C18:0), and total saturated fatty acids (SFA) having greater concentrations in the MFE group than in the LFE group. Total polyunsaturated fatty acids (PUFA) concentration was lower in the MFE group than in the LFE group. A model incorporating C14:0, C16:0, palmitoleic acid (trans9-C16:1), ante iso heptadecanoic acid plus palmitoleic acid (C17:0+trans13-C16:1), oleic acid (cis9-C18:1), cis-vaccenic acid (cis11-C18:1), petroselinic acid (cis12-C18:1), C18:0, linoleic acid (C18:2n-6), dihomo-γ-linolenic acid (C20:3n-6), cis-monounsaturated fatty acids, omega 6 PUFA, total PUFA, total SFA, other and unknown fatty acids was used to assess goodness-of-fit for RFI and showed an adjusted R 2 of 0.74. When ruminal ammonia nitrogen was added to the previous model, adjusted R 2 improved to 0.84. Our findings provide evidence that ruminal ammonia nitrogen and serum fatty acids are associated with RFI, thus suggesting that these metabolites might be helpful in identifying more feed-efficient dairy cows., (© 2025, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Objectives: To evaluate the associations between the quality of the diet and its components and microbial diversity and composition in peritoneal dialysis (PD) patients., Design and Methods: This cross-sectional study included PD patients for at least 3 months, aged 18-75 years and clinically stable. The Diet Quality Index (DQI), validated for the Brazilian population, is based on the energy density of 11 components ("sugar and sweets;" "beef, pork, and processed meat;" "refined grains and breads;" "animal fat;" "poultry, fish, and eggs;" "whole cereals, tubers, and roots;" "fruits"; "nonstarch vegetables;" "legumes and nuts;" "milk and dairy products;" and "vegetable oil"). A proportional score-based on the adequacy of the intake to Brazilian dietary guidelines-is calculated, and a final score ranged from 0 (worse) to 100 (better) is obtained. Fecal samples were collected at home, in a sterile material, kept refrigerated, and delivered to the clinic within 12 hours; α-diversity indices (Observed operational taxonomic units, Chao-1, Shannon's, Gini-Simpson's, Pielou eveness, and Faith phylogenetics) and microbial profile were determined by 16S ribosomal DNA with polymerase chain reaction-amplification and sequenced on an Illumina MiSeq platform., Results: Forty-three participants were included (53.5% men, 52.4 ± 14.1 years, body mass index: 25.9 ± 4.1 kg/m 2 , 30.2% had diabetes mellitus). DQI score was 50.5 (41.9-54.9). The lowest energy density was for the components "animal fat" and "whole cereals and breads, tubers, and roots," and the highest were for "refined grains and bread" and "beef, pork, and processed meat." Diversity indices and Enterorhabdus genus were directly associated with the energy density of the components "whole cereals and breads, tubers, and roots" and inversely with "refined grains and bread," after adjustments for age and diabetes., Conclusions: Even low, the intake of whole cereals, tubers, and roots has the potential to positively influence the microbiota profile in PD patients., (Copyright © 2025 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged 'AT-hooks' and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its 'AT-hooks' and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged 'AT-hooks.' In this paper, we demonstrate that the C-terminus significantly influences HMGA2's DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95-108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2's binding to FL814, likely by interacting with the 'AT-hooks,' as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP's binding to the 'AT-hooks.' Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Photobiomodulation (PBM) therapy, a non-thermal light therapy using nonionizing light sources, has shown therapeutic potential across diverse biological processes, including aging and age-associated diseases. In 2023, scientists from the National Institute on Aging (NIA) Intramural and Extramural programs convened a workshop on the topic of PBM to discuss various proposed mechanisms of PBM action, including the stimulation of mitochondrial cytochrome C oxidase, modulation of cell membrane transporters and receptors, and the activation of transforming growth factor-β1. They also reviewed potential therapeutic applications of PBM across a range of conditions, including cardiovascular disease, retinal disease, Parkinson's disease, and cognitive impairment. Workshop participants largely agreed that PBM holds immense potential as a safe and effective therapeutic approach for a wide range of age-related diseases and cognitive decline. While further research is needed to fully elucidate its mechanisms and optimize treatment protocols, the findings presented at the NIA workshop provide strong evidence for the continued investigation and clinical translation of this promising, inexpensive, safe technology, to aging and age-associated diseases. Here, we review the research presented and discussion held at the meeting. In addition, the text has been updated, where applicable, with recent research findings that have been made since the meeting occurred., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Transcranial infrared laser stimulation (TILS) is a noninvasive form of photobiomodulation that facilitates prefrontal energy metabolism and oxygenation, resulting in cognitive-enhancing effects. Cognitive behavioral therapy is a mainstream treatment for major depressive disorder. This is the first study to investigate whether TILS would augment the antidepressant effects of internet-based cognitive behavioral therapy., Methods: Sixty participants with major depressive disorder were given access to Deprexis, a form of internet-based cognitive behavioral therapy, for 12 weeks. After the first 2 weeks, the 40 participants who had improved at least 10% in depressive symptoms from baseline as measured by the Quick Inventory of Depressive Symptomatology-Self-Report were randomly assigned to Deprexis in combination with TILS or sham/placebo. There were no significant group differences in demographics or initial depression data., Results: There was a 43% reduction in Quick Inventory of Depressive Symptomatology-Self-Report scores in the sham group from the initial score to the week 12 score, while adding TILS as an adjunct therapy resulted in a reduction of 56%. Therefore, TILS resulted in an additional 30% reduction in Quick Inventory of Depressive Symptomatology-Self-Report scores ([56-43]/43 = 30%). Participants who received TILS to the right forehead once a week for 4 weeks showed a significantly greater reduction of depressive symptoms than participants who received sham/placebo. Participants reported no adverse effects., Conclusions: While Deprexis alone significantly reduced depression scores in the placebo control group, this beneficial effect was augmented with the addition of TILS as an adjunct therapy. Additional research that pairs neuroenhancement methods such as TILS with cognitive interventions may reveal the potential to improve treatment outcomes in depression and other psychiatric disorders., (© 2025 The Authors.)

SETD2 is known to be the unique histone methyltransferase responsible for the trimethylation of the lysine 36 of histone H3 thus generating H3K36me3. This epigenetic mark is critical for transcriptional activation and elongation, DNA repair, mRNA splicing, and DNA methylation. Recurrent SETD2-inactivating mutations and altered H3K36me3 levels are found in cancer at high frequency and numerous studies indicate that SETD2 acts as a tumor suppressor. Recently, SETD2 was further shown to methylate non-histone proteins particularly the cytoskeletal proteins tubulin and actin with subsequent impacts on cytoskeleton structure, mitosis and cell migration. Herein, we provide a review of the role of SETD2 in different cancers with special emphasis on the structural basis of the functions of this key lysine methyltransferase. Moreover, beyond the role of this enzyme in epigenetics and H3K36me3-dependent processes, we highlight the putative role of "non-epigenetic/H3K36me3" functions of SETD2 in cancer, particularly those involving the cytoskeleton., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: High-resolution anoscopy (HRA) to prevent anal cancer is complex and screening capacity is limited. Previously, we showed that DNA methylation analysis of anal high-grade squamous intraepithelial lesions (HSIL) biopsies can distinguish between HSIL with an increased cancer risk, and HSIL with a low cancer risk, in which treatment may be safely withheld. Here, we assessed the performance of methylation analysis in anal swabs to identify patients with underlying HSIL with an increased cancer risk., Methods: A cross-sectional series of paired anal swabs and biopsies of 215 persons with HIV and swabs of 19 anal cancer patients were tested for 6 methylation markers. Data were analysed by logistic regression analysis. The primary endpoint was methylation-positive biopsy HSIL (M+HSIL), indicating increased cancer risk. Test performance on swabs of methylation markers, HPV and/or cytology, as well as cancer detection and HRA referral rates were calculated., Results: Anal cancer swabs had the highest methylation levels. ZNF582, and marker panels ASCL1/ZNF582 and LHX8/ZNF582 yielded an area-under-the-curve of 0.68 to 0.70 to detect underlying M+HSIL. LHX8/ZNF582 methylation at 80% sensitivity corresponded to 43% fewer patients requiring HRA, without missing any anal cancers and detecting 79% of HPV16-positive HSIL-anal intraepithelial neoplasia grade 3. Methylation and HPV co-testing performed similarly to cytology and HPV co-testing., Conclusion: DNA methylation levels in anal swabs reflect underlying anal disease. Methylation analysis could reduce HRA referrals substantially, while maintaining a high sensitivity for M+HSIL and detecting all cancers. These results encourage screening on anal swabs to preselect patients that require HRA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Despite Brazil's recent social progress, access to health services is still unequal. This article analyzes the inter-municipal distances traveled by pregnant women to access delivery services, documenting their magnitude and relationship to socioeconomic and risk factors for over a decade in Brazil., Methods: Using data between 2007 and 2017 from the Brazilian Information System of Live Births and a matrix of inter-municipal distances, we describe the evolution of (i) the share of pregnant women that traveled across municipalities and (ii) the average distance they traveled. Next, we assess which of the previous variables explains the changes in travel distance over time. Finally, we estimate the difference in the average travel distance by individual risk factors and use regression analysis to measure the association between this distance and municipal socioeconomic determinants from the Brazilian census., Results: We observe that, on average, (i) the share of women traveling for childbirth increased, reaching 31% in 2017, and (ii) distances got longer, approaching the 60-kilometer mark by 2017. The increase in distance is mainly due to more women traveling. Nevertheless, regional disparities persist, especially between the north/inland and coastal regions. Women with high-risk pregnancies or newborns with risks such as low birth weight tend to travel longer distances. However, those residing in higher-development municipalities tend to travel shorter distances., Conclusion: Long distances remain an obstacle to accessing delivery facilities. This matter affects the most vulnerable disproportionately. Policymakers must consider the geographic accessibility of mothers when expanding birth-related services. Additionally, more research is required to understand the decision to travel and the distance effectively traveled as different accessibility facets., Competing Interests: Declarations. Ethics approval and consent to participate: We used data from open sources. The Ministry of Health of Brazil is committed to respecting ethical precepts and ensuring data privacy and security. We have confirmed that the birth-distance dataset’s fields match the publicly available Brazilian Information System of Live Births database, respecting privacy and security principles. Brazilian legislation exempts public and anonymized secondary data from ethical approval. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Despite significant advancements in the research of the pathogenesis mechanisms of Myelodysplastic Neoplasm (MDS) in recent years, there are still many gaps to fill. The advancement of metabolomics studies has led to a research booming in clarifying the impact of metabolic abnormalities during the pathogenesis of MDS. The present review primarily focuses on the dysregulated metabolic pathways, exploring the influences on the pathogenesis of MDS and their roles during the course of the disease. Furthermore, we discuss the potential of relevant metabolic pathways as therapeutic targets, along with the latest metabolic-related treatment drugs and approaches., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Daunorubicin and doxorubicin are among the most potent anti-cancer drugs and bind to DNA through intercalation. In this paper, we demonstrate that formaldehyde can efficiently and specifically conjugate daunorubicin and doxorubicin to GTP, resulting in the formation of daunorubicin-GTP-1 and doxorubicin-GTP-1 conjugates. The linkage occurs between the 2-NH 2 of guanine and the 3'-NH 2 of daunosamine. We characterized these daunorubicin/doxorubicin-GTP conjugates using various methods, including UV-Vis, fluorescence, CD, FT-IR, and mass spectrometry. Our results also indicate that these daunorubicin/doxorubicin-GTP conjugates bind to DNA via intercalation. Furthermore, we observed rapid accumulation of these conjugates in human cancer cells and observed cytotoxic effects in both doxorubicin-sensitive SK-OV-3 and doxorubicin-resistant NCI/ADR-RES cells, suggesting that these daunorubicin and doxorubicin derivatives can overcome doxorubicin resistance., (© 2024 Wiley-VCH GmbH.)

Rheumatoid arthritis (RA) is characterized by deteriorated vascular health and increased cardiovascular risk. Physical activity (PA) is recommended for cardiovascular management in RA, but evidence on the associations between objectively measured PA and vascular health markers in RA is limited. In this cross-sectional study, 82 postmenopausal women with RA (62 ± 7 yr) undertook ultrasound assessments of vascular function and structure, including brachial and superficial femoral artery (BA and SFA) flow-mediated dilation; baseline and post-hyperemia peak diameters; and carotid intima-media thickness. Participants also performed a 7-day accelerometer-based assessment of PA and sedentary behavior (SB). Fitted regression models controlled for age, body mass index, and disease activity were conducted to examine associations between vascular and PA outcomes. Regression analyses revealed that prolonged SB (bouts >60 min) and total sedentary time were inversely associated with both baseline and peak BA diameters, with each additional hour of SB resulting in decreases of 0.08-0.1 mm in these diameters ( P ≤ 0.01). Total sedentary time also showed similar negative associations with peak SFA diameters (β = -0.14 [-0.24 to -0.05], P < 0.01). Conversely, light-intensity PA and stepping time were positively associated with both baseline and peak BA diameters, with each additional hour increasing these diameters by 0.10-0.24 mm ( P ≤ 0.02). Finally, standing time was positively associated with SFA peak diameter (β = 0.11 [0.01-0.20], P = 0.02). No associations were found between moderate-to-vigorous PA and vascular outcomes. In conclusion, in patients with RA, SB was negatively, whereas light PA was positively, associated with BA and SFA diameters. These findings suggest that reducing SB and increasing PA, even at light intensities, may improve vascular health in RA. NEW & NOTEWORTHY This was the first study to investigate associations between objectively measured physical activity and markers of vascular health in rheumatoid arthritis (RA). The findings suggest that reducing sedentary behavior and increasing light or total physical activity are associated with improved vascular outcomes in RA. These results support further investigation into interventions aimed at reducing sedentary time and replacing with any type of physical activity as a potential strategy for improving cardiovascular outcomes in individuals with RA.

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States. Most recent cases of fentanyl-related overdose are linked to illicitly manufactured fentanyl and its associated extreme potency. In the present work, we describe a high-throughput analytical protocol for the screening of fentanyl analogs. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry allow for the separation and assignment of hundreds of fentanyl analogs from a single sample in a single scan. The described approach takes advantage of the recent development of data-dependent acquisition and data-independent acquisition using parallel accumulation in the mobility trap followed by sequential fragmentation using collision-induced dissociation. The fentanyl analogs are confidently assigned based on their retention time, mobility, and MS fragmentation pattern.

Conventional ameloblastoma presents infiltrative behavior and its treatment ranges from enucleation combined with adjuvant therapies to marginal/segmental resection. The purpose of this study is to present a cohort of twenty-four patients with ameloblastoma treated in the same institution after marginal/segmental resection for the past 21 years. All cases had diagnosis confirmation by incisional biopsy. Patients with an unconfirmed diagnosis and missing follow-up information were excluded. Data were categorized into clinicopathological, surgical and recurrence aspects. Thirteen patients were females (54%). The mean age was 40.2 years. Mandible was the most affected site (91%). The mean length of the lesions was 4.10 cm (± 2.06) and the multilocular aspect was predominant (83%). Root resorption (37.5%), tooth displacement (45.8%) and cortical perforation (45.8%) were noticed. Histologically, most of the cases were follicular (n = 19,79%). Microscopic analysis showed positive margins in four cases. Patients were treated by marginal (n = 19) and segmental (n = 5) resections. Recurrence occurred in two cases (8.33%). Both primary and recurrent ameloblastomas were treated through marginal resections and no recurrence was observed during the past 9 and 5 years after the last intervention, respectively. The overall mean follow-up was 79.25 months and patients are still monitored over these years. Marginal/segmental resection of conventional ameloblastoma is associated with a low recurrence rate., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Previously, we showed that omega-3 polyunsaturated fatty acid n-3 (PUFA) supplementation improved the performance of postpartum rats in the shuttle box escape test (SBET)., Methods: The brains of these rats were used in the current study which examined brain cytochrome c oxidase (CCO) activity in white matter bundles and 39 regions spanning sensorimotor, limbic, and cognitive areas to determine the effects of n-3 PUFAs on neural metabolic capacity and network interactions., Results: We found that n-3 PUFA supplementation decreased CCO activity in white matter bundles, deep and superficial areas within the inferior colliculus, the anterior and barrel field regions of the primary somatic sensorimotor cortex, the secondary somatic sensorimotor cortex, the lateral, anterior regions of the secondary visual cortex and the ventral posterior nucleus of the thalamus, and the medial nucleus of the amygdala. Structural equation modeling revealed that animals consuming diets without n-3 PUFAs exhibited fewer inter-regional interactions when compared to those fed diets with n-3 PUFAs. Without n-3 PUFAs, inter-regional interactions were observed between the posterior cingulate cortex and amygdala as well as among amygdala subregions. With n-3 PUFAs, more inter-regional interactions were observed, particularly between regions associated with fear memory processing and escape. Correlations between regional CCO activity and SBET behavior were observed in rats lacking dietary n-3 PUFAs but not in those supplemented with these nutrients., Discussion: In conclusion, consumption of n-3 PUFAs results in reduced CCO activity in white matter bundles and sensorimotor regions, reflecting more efficient neurotransmission, and an increase in inter-regional interactions, facilitating escape from footshock., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rivers, Farber, Heath, Gonzales, Barrett, Gonzalez-Lima and Lane.)

Graphene oxide (GO) undergoes multiple transformations when introduced to biological and environmental media. GO surface favors the adsorption of biomolecules through different types of interaction mechanisms, modulating the biological effects of the material. In this study, we investigated the interaction of GO with tannic acid (TA) and its consequences for GO toxicity. We focused on understanding how TA interacts with GO, its impact on the material surface chemistry, colloidal stability, as well as, toxicity and biodistribution using the Caenorhabditis elegans model. Employing computational modeling, including reactive classical molecular dynamics and ab initio calculations, we reveal that TA preferentially binds to the most reactive sites on GO surfaces via the oxygen-containing groups or the carbon matrix; van der Waals interaction forces dominate the binding energy. TA exhibits a dose-dependent mitigating effect on the toxicity of GO, which can be attributed not only to the surface interactions between the molecule and the material but also to the inherent biological properties of TA in C. elegans . Our findings contribute to a deeper understanding of GO's environmental behavior and toxicity and highlight the potential of tannic acid for the synthesis and surface functionalization of graphene-based nanomaterials, offering insights into safer nanotechnology development., Competing Interests: There are no conflicts to declare., (Copyright © 2024, Petry et al.)

The "Histone Code" is comprised of specific types and positions of post-translational modifications (PTMs) which produce biological signals for gene regulation and have potential as biomarkers for medical diagnostics. Previous work has shown that electron-based fragmentation improves the sequence coverage and confidence of labile PTM position assignment. Here, we evaluated two derivatization methods (e.g., irreversible - propionylation and reversible-citraconylation) for bottom-up analysis of histone H4 4-17 proteoforms using online liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), and electron-based dissociation (ExD) in tandem with mass spectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF MS/MS based on a Bruker Impact and a commercial μLC-EAD-ToF MS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4 4-17 0-4ac positional isomer standards showed that they can be resolved in their endogenous form, while positional isomers cannot be fully resolved in their propionylated form; online LC-ExD-MS/MS provided high sequence coverage (>90%) for all H4 4-17 (0-4ac) proteoforms in both instrumental platforms. When applied to model cancer cells treated with a histone deacetylase inhibitor (HeLa + HDACi), both derivatization methods and platforms detected and confirmed H4 4-17 (0-4ac) proteolytic peptides based on their fragmentation pattern. Moreover, a larger number of HeLa + HDACi H4 4-17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF MS/MS due to the positional isomer preseparation in the LC-TIMS domain of citraconylated H4 4-17 (0-4ac) peptides.

Photoaffinity labeling is a widely used technique for studying ligand-protein and protein-protein interactions. Traditional photoaffinity labels utilize nonspecific C-H bond insertion reactions mediated by a highly reactive intermediate. Despite being the most widely used photoaffinity labels, diazirines exhibit limited compatibility with downstream organic reactions and suffer from storage stability concerns. This study introduces oxadiazolines as innovative and complementary photoactivatable labels for addition to the toolbox and demonstrates their application in vitro and through in cellulo labeling experiments. Oxadiazolines can be easily synthesized from ketone moieties and cleaved with 302-330 nm light to cleanly liberate a diazo reactive moiety that can covalently modify nucleophilic amino acid residues. Notably, oxadiazolines are compatible with various organic reaction conditions and functional groups, allowing for the exploration of a large chemical space. Several known inhibitors featuring the oxadiazoline functionality were prepared without affecting their binding affinity. Furthermore, we confirmed the ability of oxadiazolines to form covalent bonds with proteins upon UV-irradiation, both in vitro and in cellulo , yielding comparable results to those of the matched diazirine compounds.

The molecular level characterization of complex mixtures remains an analytical challenge. We have shown that the integration of complementary, high-resolution, gas-phase separations allows for chemical formula level isomeric content description. In the current work, we revisited the current challenges associated with the analysis of dissolved organic matter using high-resolution trapped ion mobility separation (TIMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In particular, we evaluated the separation capabilities provided by TIMS-MS compared to MS alone, the use of ICR complementary data acquisition (DAQ) systems and transient processing strategies, ICR cell geometries (e.g., Infinity cell vs harmonized cell), and magnetic field strengths (7 T vs 9.4 T vs 21 T) for the case of a Harney River DOM sample. Results showed that the external high-performance DAQ enables direct representation of mass spectra in absorption mode FT (aFT), doubling the MS resolution compared to the default magnitude mode FT (mFT). Changes between half- vs full-apodization result in greater MS signal/noise vs superior MS resolving power (RP); in the case of DOM analysis, a 45% increase in assigned formulas is observed when employing the DAQ half (Kaiser-type)-apodization window and aFT when compared to the default instrument mFT. Results showed the advantages of reprocessing 2D-TIMS-FT-ICR MS data with higher RP and magnetic field chemical formulas generated list acquired (e.g., 21 T led to a 24% increase in isomers reported) or the implementation of alternative strategies.

We report experimental investigations of spin-to-charge current conversion and charge transfer (CT) dynamics at the interface of the graphene/WS 2 van der Waals heterostructure. Pure spin current was produced by the spin precession in the microwave-driven ferromagnetic resonance of a permalloy film (Py=Ni 81 Fe 19 ) and injected into the graphene/WS 2 heterostructure through a spin pumping process. The observed spin-to-charge current conversion in the heterostructure is attributed to the inverse Rashba-Edelstein effect (IREE) at the graphene/WS 2 interface. Interfacial CT dynamics in this heterostructure was investigated based on the framework of the core-hole clock (CHC) approach. The results obtained from spin pumping and CHC studies show that the spin-to-charge current conversion and charge transfer processes are more efficient in the graphene/WS 2 heterostructure compared to isolated WS 2 and graphene films. The results show that the presence of WS 2 flakes improves the current conversion efficiency. These experimental results are corroborated by density functional theory (DFT) calculations, which reveal (i) Rashba spin-orbit splitting of graphene orbitals and (ii) electronic coupling between graphene and WS 2 orbitals. This study provides valuable insights for optimizing the design and performance of spintronic devices.

We discuss the short-time behavior of the majority vote dynamics on scale-free networks at the critical threshold. A heterogeneous mean-field theory on the critical short-time behavior of the majority-vote model on scale-free networks is introduced. In addition, the heterogeneous mean-field predictions are compared with extensive Monte Carlo simulations of the short-time dependencies of the order parameter and the susceptibility. Closed expressions of the dynamical exponent z and the time correlation exponent ν&#95;{∥} are obtained. The short-time scaling is compatible with a nonuniversal critical behavior for 5/2<γ<7/2. However, for γ≥7/2, we have the mean-field Ising criticality with additional logarithmic corrections for γ=7/2, the same as the stationary scaling.

This pilot study aimed to investigate the relation between cardio-respiratory parameters derived from Central Venous Pressure (CVP) waveform and Extubation Failure (EF) in mechanically ventilated ICU patients during post-extubation period. This study also proposes a new methodology for analysing these parameters during rest/sleep periods to try to improve the identification of EF. We conducted a prospective observational study, computing CVP-derived parameters including breathing effort, spectral analyses, and entropy in twenty critically ill patients post-extubation. The Dynamic Warping Index (DWi) was calculated from the respiratory component extracted from the CVP signal to identify rest/sleep states. The obtained parameters from EF patients and patients without EF were compared both during arbitrary periods and during reduced DWi (rest/sleep). We have analysed data from twenty patients of which nine experienced EF. Our findings may suggest significantly increased respiratory effort in EF patients compared to those successfully extubated. Our study also suggests the occurrence of significant change in the frequency dispersion of the cardiac signal component. We also identified a possible improvement in the differentiation between the two groups of patients when assessed during rest/sleep states. Although with caveats regarding the sample size, the results of this pilot study may suggest that CVP-derived cardio-respiratory parameters are valuable for monitoring respiratory failure during post-extubation, which could aid in managing non-invasive interventions and possibly reduce the incidence of EF. Our findings also indicate the possible importance of considering sleep/rest state when assessing cardio-respiratory parameters, which could enhance respiratory failure detection/monitoring., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Fucosylated carbohydrate antigens play critical roles in physiology and pathology with function linked to their structural details. However, the separation and structural characterization of isomeric fucosylated epitopes remain challenging analytically. Here, we report for the first time the influence of alkali metal cations (Li + , Na + , K + , Rb + , and Cs + ) and halogen anions (Cl - , Br - , and I - ) on the gas-phase conformational landscapes of common fucosylated trisaccharides (Lewis A, X, and H types 1 and 2) and tetrasaccharides (Lewis B and Y) using trapped ion mobility spectrometry coupled to mass spectrometry and theoretical calculations. Inspection of the mobility profiles of individual standards showed a dependence on the number of mobility bands with the oligosaccharide and the alkali metal and halogen; collision cross sections are reported for all of the observed species. Results showed that trisaccharides (Lewis A, X, and H types 1 and 2) can be best mobility resolved in the positive mode using the [M + Li] + molecular ion form (baseline resolution r ≈ 2.88 between Lewis X and A); tetrasaccharides can be best mobility resolved in the negative mode using the [M + I] - molecular ion form (baseline separation r ≈ 1.35 between Lewis B and Y). The correlation between the number of oligosaccharide conformers as a function of the molecular ion adduct was studied using density functional theory. Theoretical calculations revealed that smaller cations can form more stable structures based on the number of coordinations, while larger cations induced greater oligosaccharide reorganizations; candidate structures are proposed to better understand the gas-phase oligosaccharide rearrangement trends. Inspection of the candidate structures suggests that the interplay between ion size/charge density and molecular structure dictated the conformational preferences and, consequently, the number of mobility bands and the mobility separation across isomers. This work provides a fundamental understanding of the gas-phase structural dynamics of fucosylated oligosaccharides and their interaction with alkali metals and halogens.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to native ion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary structural information about the conformational dynamics of biological molecules. In the present work, the solution-stable isotope labeling (SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem with top-down electron capture dissociation (ECD) is illustrated for the structural characterization of the solution native states of ubiquitin. Four different ubiquitin electrospray solution conditions: (i) single-tip nondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip SIL-deuterated, and (iv) theta tip for online SIL H/D back exchange (HDbX), were investigated to assess the H/D exchange reactivities of native ubiquitin. The combination of TIMS and ECD in a q-ToF MS instrument allowed for additional inspection of gas-phase HDbX added by top-down fragmentation, revealing the exposed and protected residues with limited scrambling effects (e.g., intramolecular H/D migration). A native charge state distribution (5+ to 7+) and TIMS profiles were observed under the single-tip nondeuterated solution conditions. Mass shift distributions of ∼40, ∼104, and ∼87D were observed when incorporating deuterium for online SIL HDX, SIL HDX, and online SIL HDbX, respectively, while retaining similar conformational states. ECD fragmentation allowed for the localization of the deuterated labeled residues of the peptide fragments, with a sequence coverage of ∼90%, for each of the ubiquitin solution condition. Changes in the TIMS trapping time settings (∼70 to ∼795 ms) were used to determine the H/D back exchange dynamics of native ubiquitin. HDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue C-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63 residues, indicating that these regions are the most exposed area (less protected hydrogens) of ubiquitin as compared to the rest of the core residues that adopt a compact β-grasp fold (protected hydrogens), which was consistent with the accessible surface area of ubiquitin. The present data highlight for the first time consistency between the solution HDX and gas-phase HDbX-TIMS data for native studies.

Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 5 2 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 6 1 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature ( T ∼20-95 °C). An increase in the protein charge states and collision cross sections (∼2750-8750 Å 2 and ∼3250-15,385 Å 2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.

Identified as a potential reference pathogen by the WHO Guidelines for Drinking-Water Quality, Rotavirus (RV) is among the main enteric viruses that cause waterborne diseases. The aim of this study was to identify and correlate the presence of RV in collective and individual water sources of rural communities in the state of Goiás, within the seasons in which the collections were made (rainy and dry seasons). For this, 86 water samples in the dry period and 160 samples in the rainy period were collected. Concentration of water samples, extraction of viral genetic material and molecular tests were performed. When analyzing the presence of RV in the samples, taking into consideration the period studied, RV was found to be more prevalent in the dry season (54.7%) than in the rainy season (20%), showing a strong statistical association with the dry season ( p -value < 0.001). The presence of pathogenic microorganisms in water is a public risk issue, enabling the emergence of outbreaks, endemics and epidemics. In the present research, there was an association between the presence of Rotavirus and the dry period of the year when compared to the rainy period.

Background: Multiple sclerosis (MS) is an inflammatory chronic autoimmune and neurodegenerative disorder of the brain and spinal cord, resulting in loss of motor, sensorial, and cognitive function. Among the non-pharmacological interventions for several brain conditions, photobiomodulation (PBM) has gained attention in medical society for its neuroprotective effects. We systematically reviewed the effects of PBM on MS., Methods: We conducted a systematic search on the bibliographic databases (PubMed and ScienceDirect) with the keywords based on MeSH terms: PBM, low-level laser therapy, multiple sclerosis, autoimmune encephalomyelitis, demyelination, and progressive multiple sclerosis. Data search was limited from 2012 to July 2024. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The initial systematic search identified 126 articles. Of these, 68 articles were removed by duplicity and 50 by screening. Thus, 8 studies satisfied the inclusion criteria., Results: The reviewed studies showed that PBM modulates brain markers linked to inflammation, oxidative stress, and apoptosis. Improvements in motor, sensorial, and cognitive functions in MS patients were also observed after PBM therapy. No study reported adverse effects of PBM., Conclusion: These findings suggest the potential of PBM as a promising non-pharmacological intervention for the management of MS, although further research is needed to standardize PBM protocols and assess its long-term effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oliveira de Andrade Filho, Amarante, Gonzalez-Lima, Gomes da Silva and Cardoso.)

The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K 0 ) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me 1-3 ) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

An exome sequencing strategy employed to identify pathogenic variants in patients with pediatric-onset systemic lupus or Evans syndrome resulted in the discovery of six novel monoallelic mutations in PTPN2. PTPN2 is a phosphatase that acts as an essential negative regulator of the JAK/STAT pathways. All mutations led to a loss of PTPN2 regulatory function as evidenced by in vitro assays and by hyperproliferation of patients' T cells. Furthermore, patients exhibited high serum levels of inflammatory cytokines, mimicking the profile observed in individuals with gain-of-function mutations in STAT factors. Flow cytometry analysis of patients' blood cells revealed typical alterations associated with autoimmunity and all patients presented with autoantibodies. These findings further supported the notion that a loss of function in negative regulators of cytokine pathways can lead to a broad spectrum of autoimmune manifestations and that PTPN2 along with SOCS1 haploinsufficiency constitute a new group of monogenic autoimmune diseases that can benefit from targeted therapy., (© 2024 Jeanpierre et al.)

The main objective of this study was to evaluate the effect of ceftiofur on metritis cure, milk yield, reproductive performance, and culling up to 300 DIM. The secondary objective was to evaluate the effect of metritis cure at 5 (early cure [ECURE]) and 14 (late cure [LCURE]) days after diagnosis on milk production, reproduction, and culling. A total of 422 Holstein cows diagnosed with metritis from 4 herds located in Texas, California, and Florida were enrolled in a randomized clinical trial. Cows diagnosed with metritis (fetid, watery, reddish or brownish uterine discharge) were blocked by herd and parity and were randomly allocated to receive systemic administration of ceftiofur (CEF) or to remain untreated (CON). In addition, 399 nonmetritic cows (NMET) were included for comparison purposes. Metritis cure was evaluated at 5 and 14 d after diagnosis and was defined as the absence of metritis clinical signs. Logistic regression models were fitted to the data to assess the effect of treatment on metritis cure. Milk yield was analyzed using a mixed linear model, while logistic regression, Cox proportional hazard, and Kaplan-Meier survival analysis models were fitted to culling and reproduction data. Cows treated with CEF had 1.86 (95% CI: 1.22-2.81) and 1.68 (95% CI: 1.02-2.75) greater odds of being cured than CON cows at 5 and 14 d after diagnosis, respectively. No effect of CEF was observed for milk yield; however, NMET cows had greater milk yield compared with metritic cows (CEF = 36.0, 95% CI = 33.8-38.1; CON = 36.1, 95% CI = 33.9-38.2; NMET = 36.9 kg/d, 95% CI = 34.8-39.4). Likewise, no effect of CEF was observed on reproductive performance and culling. Nonetheless, the likelihood of conceiving for NMET cows was 1.72 (95% CI = 1.41-2.12) and 1.64 (95% CI = 1.33-2.00) times greater than for CEF and CON cows, respectively. Ceftiofur-treated and CON cows had 2.93 (95% CI = 1.90-4.51) and 2.37 (95% CI = 1.51-3.71) greater hazard of culling compared with NMET, respectively. Regardless of treatment, no differences between ECURE and LCURE were observed on milk yield, reproduction, and culling throughout the entire lactation, but cows that cured at 5 or 14 d after diagnosis had greater milk production in the first 60 DIM compared with cows that did not cure (NCURE). Cows in ECURE and LCURE also had a 1.59 (95% CI = 1.16-2.16) and 1.49 (95% CI = 1.08-2.05) greater hazard of pregnancy and 0.43 (95% CI = 0.26-0.71) and 0.56 (95% CI = 0.34-0.92) hazard of culling compared with NCURE. Ceftiofur therapy increased metritis cure, but benefits to productivity and longevity were not observed. Also, cows that fail to cure have impaired lactation performance, but no differences regarding timing of cure were observed., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (<1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

The initial ovulatory response during synchronization programs is often low in dairy heifers, largely due to follicular dynamics and hormonal dynamics. Specifically, the progesterone (P4) concentration at the time of the first GnRH treatment in a breeding program can influence the LH response, often resulting in a suboptimal ovulatory response. The objective of this study was to determine the effect of the highest label dose 200 μg (100 μg vs. 200 μg) of GnRH (50 μg of gonadorelin hydrochloride per mL; Factrel, Zoetis Inc. Madison, NJ) at the first GnRH of a 6-d CO-Synch plus P4 device program on ovulatory response and pregnancy per AI (P/AI) in first service in Holstein heifers. A total of 1,308 Holstein heifers were randomly allocated at the beginning of a 6-d CO-Synch program at day 0 to receive either i.m. treatment of 100 μg (2CC, n = 655) or 200 μg (4CC, n = 653) of GnRH. Also, at d 0, heifers received an intravaginal insert with 1.38 g of P4 (Eazi-Breed CIDR Cattle Insert, Zoetis Inc.). On day 6, the insert was removed, and i.m. treatment of 25 mg of PGF 2α (12.5 mg of dinoprost tromethamine/mL; Lutalyse HighCon Injection, Zoetis Inc.) was administered. On d 7, a second i.m. treatment of 25 mg of PGF 2α was given, followed on d 9 by concurrent i.m. treatment of 100 μg of GnRH, and timed AI. A subset of 396 heifers had their ovaries scanned to evaluate ovulatory response, and blood samples were collected to measure the serum concentration of P4 at d 0 and d 6 of the study. The P4 concentrations at d 0 were categorized as low (≤3 ng/mL) or high (>3 ng/mL). The ovulatory response was greater for heifers receiving 4CC than 2CC at d 0 (54.7% vs. 42.8%). The ovulatory response was greater for low P4 than high P4 at d 0 (54.3% vs. 37.8%). However, we did not observe an interaction between treatment and P4 concentrations (low P4 2CC = 48.6% vs. high P4 2CC = 30.0%; low P4 4CC = 60.0% vs. high P4 4CC = 45.5%). The receiver operating characteristic curve analysis indicated that P4 concentrations at d 0 treatment could predict the ovulatory response, although the area under the curve was only 0.6. As expected, heifers that ovulated had increased P/AI (no = 55.6% vs. yes = 67.7%); however, we found no effect of treatment on P/AI (2CC = 63.3% vs. 4CC = 59.6%), and no interactions between treatment and ovulation and treatment and P4 (high vs. low) for pregnancy outcomes. In summary, P4 concentration and increasing the dose of GnRH at d 0 positively affected ovulatory response in Holstein heifers. However, there was no interaction between treatment and P4 on ovulation and no subsequent impact of GnRH dose on P/AI., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated-health monitoring device (AHMD) from -21 ± 3 to 60 ± 3 d relative to calving to monitor rumination time and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 d in milk (DIM). Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD), were randomly allocated to 1 of 2 treatments: control (CON; n = 62), no treatment at the time of metritis diagnosis (d 0); or ceftiofur (CEF; n = 70), subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC, and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and DIM at metritis diagnosis) and behavior data (i.e., daily averages of rumination time, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with P-values ≤0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, with sensitivity (Se) of 89% and specificity (Sp) of 77%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, with Se of 93% and Sp of 87%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Our objective was to evaluate the accuracy of predictive models for metritis spontaneous cure (SC) and cure among ceftiofur-treated cows using farm-collected data only, and with the addition of hemogram variables and circulating concentration of metabolites, minerals, and biomarkers (BM) of inflammation measured at time of diagnosis. Data related to parity, calving-related issues, BCS, rectal temperature, and DIM at metritis diagnosis were collected from a randomized clinical trial that included 422 metritic cows from 4 herds in Texas, California, and Florida. Metritis was defined as the presence of red-brownish, watery, and fetid vaginal discharge, and cure was defined as the absence of metritis 14 d after initial diagnosis. Cows were randomly allocated to receive systemic ceftiofur therapy (2 subcutaneous doses of 6.6 mg/kg of ceftiofur crystalline-free acid on the day of diagnosis and 3 d later; CEF) or to remain untreated (control). At enrollment (day of metritis diagnosis), blood samples were collected and submitted to complete blood count (CBC) and processed for the measurement of 13 minerals and BM of metabolism and inflammation. Univariable analysis to evaluate the association of farm-collected data and blood-assessed variables with metritis cure were performed, and variables with P ≤ 0.20 were offered to multivariable logistic regression models and retained if P ≤ 0.15. The areas under the curve for models predicting SC using farm data only and farm + BM were 0.70 and 0.76, respectively. Complete blood count variables were not retained in the models for SC. For models predicting cure among CEF cows, the area under the curve was 0.75, 0.77, 0.80, and 0.80 for models using farm data only, farm + CBC, farm + BM, and farm + CBC + BM, respectively. Predictive models of metritis cure had fair accuracy, with SC models being less accurate than models predictive of cure among CEF cows. Additionally, adding BM variables marginally improved the accuracy of models using farm collected data, and CBC data did not improve the accuracy of predictive models., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Approximately 60% of individuals with cystic fibrosis (CF) are affected by Aspergillus fumigatus infection. This condition is correlated with a decline in lung function and is identified as an independent risk factor contributing to hospital admissions among CF patients. This study investigates the dynamic interplay of A. fumigatus within the context of CF patients, tracing its evolution over time, with a specific emphasis on colonization dynamics., Methods: An analysis was conducted on 83 sequential A. fumigatus isolates derived from sputum samples of six patients receiving care at a renowned CF hospital in Brazil. Employing microsatellite genotyping techniques, alongside an investigation into cyp 51A gene mutations, this research sheds light on the genetic variations, colonization, and resistance of A. fumigatus within the CF respiratory environment., Results: Our research findings indicate that CF patients can harbor A. fumigatus strains from the same clonal complexes for prolonged periods. Additionally, we identified that clinical isolates have the potential to spread among patients in the same healthcare facility, evidencing hospital contamination. Two patients who underwent long-term Itraconazole treatment did not show phenotypic resistance. However, one of these patients exhibited mutations in the cyp 51A gene, indicating the need to monitor resistance to azoles in these patients colonized for long periods by A. fumigatus . We also observed co-colonization or co-infection involving multiple genotypes in all patients over time., Conclusion: This comprehensive examination offers valuable insights into the pathogenesis of A. fumigatus infections in CF patients, potentially shaping future therapeutic strategies and management approaches. This enhanced understanding contributes to our knowledge of A. fumigatus impact on disease progression in individuals with cystic fibrosis. Additionally, the study provides evidence of cross-contamination among patients undergoing treatment at the same hospital.

The Brazilian Legal Amazon is an extensive territory in which different factors influence the dynamics of forest fires. Currently, the Brazilian government has two tools in the public domain and free of charge, PRODES and BDQueimadas, to monitor and make decisions to combat deforestation and forest fires. This work aimed to evaluate and correlate the forest fire alerts and deforestation in the Amazon Forest in the state of Pará. The analyses were based on carrying out a diagnosis of forest fires and deforestation; the behavior of forest fires and deforestation over the last twenty years; the statistical relationship between deforestation and forest fires and their spatialization. This work identified that Pará is the state in the Legal Amazon with the highest occurrence of forest fires and deforestation. Deforestation in the four-year period Jan/2003-Dec/2006 showed a higher rate compared to the four-year periods Jan/2011-Dec/2018. A high correlation was found between forest fire alerts and increases in deforestation. There is a spatial relationship between cities with greater increases in deforestation and high numbers of fire alerts. In relation to the occurrence of forest fires and deforestation, the south of the state was the most critical region and the north had lower rates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Furtado Lima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The chromenopyridine scaffold represents an important class of heterocyclic compounds exhibiting a broad spectrum of biological properties. This review describes novel and efficient procedures for the synthesis of this scaffold. Herein, several methods were detailed and grouped according to their starting material (e.g., salicylaldehydes, chromones, chromanones and coumarins) and respective biological activity, when reported. This review highlights the potential of the reported synthetic strategies for preparing chromenopyridine derivatives with promising biological activity, paving the way for further developments in drug discovery.

Omphalocele is a malformation of the abdominal wall varying in size and located at the base of the umbilical cord. Meckel's diverticulum is the most common congenital malformation of the gastrointestinal tract with an increased prevalence in newborns with congenital malformations of the umbilicus and gastrointestinal tract. The association between Meckel's diverticulum and omphalocele has been described in rare cases. We present the case of a newborn diagnosed with both entities., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Valente et al.)

Background: Individuals with minor ischaemic stroke and intracranial occlusion are at increased risk of poor outcomes. Intravenous thrombolysis with tenecteplase might improve outcomes in this population. We aimed to test the superiority of intravenous tenecteplase over non-thrombolytic standard of care in patients with minor ischaemic stroke and intracranial occlusion or focal perfusion abnormality., Methods: In this multicentre, prospective, parallel group, open label with blinded outcome assessment, randomised controlled trial, adult patients (aged ≥18 years) were included at 48 hospitals in Australia, Austria, Brazil, Canada, Finland, Ireland, New Zealand, Singapore, Spain, and the UK. Eligible patients with minor acute ischaemic stroke (National Institutes of Health Stroke Scale score 0-5) and intracranial occlusion or focal perfusion abnormality were enrolled within 12 h from stroke onset. Participants were randomly assigned (1:1), using a minimal sufficient balance algorithm to intravenous tenecteplase (0·25 mg/kg) or non-thrombolytic standard of care (control). Primary outcome was a return to baseline functioning on pre-morbid modified Rankin Scale score in the intention-to-treat (ITT) population (all patients randomly assigned to a treatment group and who did not withdraw consent to participate) assessed at 90 days. Safety outcomes were reported in the ITT population and included symptomatic intracranial haemorrhage and death. This trial is registered with ClinicalTrials.gov, NCT02398656, and is closed to accrual., Findings: The trial was stopped early for futility. Between April 27, 2015, and Jan 19, 2024, 886 patients were enrolled; 369 (42%) were female and 517 (58%) were male. 454 (51%) were assigned to control and 432 (49%) to intravenous tenecteplase. The primary outcome occurred in 338 (75%) of 452 patients in the control group and 309 (72%) of 432 in the tenecteplase group (risk ratio [RR] 0·96, 95% CI 0·88-1·04, p=0·29). More patients died in the tenecteplase group (20 deaths [5%]) than in the control group (five deaths [1%]; adjusted hazard ratio 3·8; 95% CI 1·4-10·2, p=0·0085). There were eight (2%) symptomatic intracranial haemorrhages in the tenecteplase group versus two (<1%) in the control group (RR 4·2; 95% CI 0·9-19·7, p=0·059)., Interpretation: There was no benefit and possible harm from treatment with intravenous tenecteplase. Patients with minor stroke and intracranial occlusion should not be routinely treated with intravenous thrombolysis., Funding: Heart and Stroke Foundation of Canada, Canadian Institutes of Health Research, and the British Heart Foundation., Competing Interests: Declaration of interests SBC received grant funding from the Canadian Institutes of Health Research (CIHR), Heart and Stroke Foundation of Canada (HSFC), and the British Heart Foundation (BHF) to complete the TEMPO-2 study. Boehringer Ingelheim provided off-the-shelf study drug (tenecteplase) for the study. JFA has received public research grants from the Spanish Ministry of Science, the regional health department, the European Commission, and a private research grant from AstraZeneca. He has received consultant or speaker fees from Pfizer-BMS, Medtronic, and Amgen, and travel support from Daiichi-Sankyo. KSB reports speaker fees from Boehringer Ingelheim and AstraZeneca. LC has received consulting or speakers fees from Roche. JF reports speaker fees from AstraZeneca, Bayer, BMS, Boehringer Ingelheim, Pfizer, Sankyo, and Daiichi. AG reports a grant from Microvention; speaker fees from Alexion, Biogen, and Servier Canada; and stock options for SnapDx and Collavidence. DG reports honoraria from Boehringer Ingelheim, Ipsen, and Eisai. MG reports grant funding from Medtronic and Cerenovus and consulting fees from Mentice, CSL Behring, MicroVention, and Medtronic. TSF participated in an advisory board for Roche. FL reports speaker fees and travel support from Boehringer Ingelheim. BKM has been paid honoraria from Boehringer Ingelheim and Roche. MM has received honoraria from Boehringer Ingelheim for participation at an advisory board on the approval of tenecteplase for treatment of acute ischaemic stroke. SOM has received speaker fees from Boehringer, Medtronic, Penumbra, Bayer, Pfizer, Novartis, Novo Nordisk, Servier, and Daiichi Sankyo. KWM reports grant funding from the BHF, and the Stroke Association; consultancy fees from Boehringer Ingelheim, Biogen, IschaemaView; lecture fees from Boehringer Ingelheim, IschaemaView, and Brainomix; and non-financial support (drug supply for ATTEST-2 trial) from Boehringer Ingelheim. MWP is on Boehringer Ingelheim Advisory Board for Metalyse in stroke. AP received speaker fees from Boehringer Ingelheim and travel support from AstraZeneca for attending a meeting. OMP-N has received speaker fees from Boehringer Ingelheim and AstraZeneca. RS is part funded by the UCLH Biomedical Research Centre. DS reports an unrestricted educational grant from Boehringer Ingelheim. MIW reports sponsorship or financial assistance to support attendance at scientific meetings from Boehringer Ingelheim, Bristol Myers Squibb, Daiichi Sankyo UK, and Bayer; honoraria for lectures given at education meetings from Boehringer Ingelheim, Bristol Myers Squibb, and AstraZeneca; and payment for participation in advisory panels from Boehringer Ingelheim and Daiichi Sankyo UK. DJW is the Co-principal Investigator for Health Research Board of Ireland, Award–Improving Pathways for Acute Stroke and Rehabilitation. AYXY reports salary support from the HSFC. MDH reports grant funding from CIHR, HSFC, Alberta Innovates, and study drug from Boehringer Ingelheim; grants from NoNo and Medtronic; consulting fees from Sun Pharma Brainsgate; and a DSMB member role on many stroke clinical trials., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)