bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of Intrathecal 0.75% Hyperbaric Ropivacaine versus 0.5% Hyperbaric Bupivacaine for Elective Inguinal and Perineal Surgery: A Randomised Double-blinded Clinical Study
Authors: Shah Bhavini , Mahanta Dipanjali
Published: January 2025

Introduction: Ropivacaine, an amide local anaesthesia, often considered safer alternative to bupivacaine. Bupivacaine, a longer-acting agent that provides dense motor blockade, is known to carry a higher risk of re-entrant arrhythmias and cardiac depression with accidental intravascular injection. The shorter duration of action, coupled with profound sensory block and comparatively less side-effects, makes ropivacaine useful in tackling cases of day care surgery.

Aim: To compare the efficacy of hyperbaric ropivacaine versus bupivacaine for elective inguinal and perineal surgery under spinal anaesthesia in terms of time of sensory and motor block, time taken to reach peak T8 level sensory block, total duration of both sensory and motor block, time of first micturition, and any associated side-effects.

Materials and Methods: The present randomised double-blind clinical study, was carried out in the Department of Anaesthesia, Dr. D. Y Patil Medical College, Hospital and Research Centre (tertiary healthcare centre), Pune, Maharashtra, India, from March 2023 to August 2023. Study involved 30 patients aged 18-60, classified as American Society of Anaesthesiologists (ASA) grade I and II. Patients were randomly assigned to group RH (receiving hyperbaric ropivacaine) or group BH (receiving hyperbaric bupivacaine). The study assessed for onset and duration of sensory block at T-8 dermatome. The study also assessed motor block onset and duration using modified Bromage scale, observing postoperative micturition, intraoperative haemodynamic changes and adverse effects. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software version 16.0, with a p-value of <0.05 considered significant.

Results: Demographic profile in terms of age {Mean±Standard Deviation (SD) in group RH: 42.93±14.73 years vs group BH: 39.06±11.94 years), gender distribution, type of surgery performed and its duration (group RH: 85.67±39.5 minutes vs group BH: 98.4±32.8 minutes) was not significant. The onset of sensory block (2.09±0.69 min) and motor block (3.52±0.66 min), the time taken to reach T8 level (3.40±0.93 min), was faster in the hyperbaric ropivacaine group. Similarly, the duration of motor block (261.93±73.21 min), time taken for the recession of the sensory block to S2 (351.4±66.78 min), and the recession of the motor block to Bromage scale 0 (291.93±64.93 min) were faster in ropivacaine group. The time taken for the passage of first micturition post-subarachnoid block was earlier (338.73±31.29 min) in ropivacaine group.

Conclusion: A 0.75% hyperbaric ropivacaine is more efficacious, with faster onset of sensory and motor blockade, relatively quick recovery profile, and an earlier time of first micturition in comparison to 0.5% hyperbaric bupivacaine.
Shaping Minds and Societies: The Legal History of Education in China
Authors: Ruhal Mir , Linxin Qu , Liberte Efa Efa Ange , Khan Abdul Basit , Mafruhah Syarifatul , Zaheer Sajawal
Published: January 2025

This paper seeks to present a legal study of the educational systems in China as well as to establish legal developments of laws and policies and its reforms within China’s educational systems. Starting with the imperial education laws based on the Confucianism which largely stressed the imperial order and helped to select bureaucratic officials, the discussion proceed with the education laws of the republican era, with founding of the People’s Republic of China (PRC) in 1949, and with the modern legal changes for education aimed at extending, equalizing and enhancing education. In the case of education, laws were passed and implemented mainly as social engineering policies to fight illiteracy; to incite an increased enrolment of children into primary schools, and to address inequity. Another major step was made in 1986 when the National People’s Congress adopted the Education Law regulating compulsory education as the right of every citizen, establishing nine year compulsory education, and placing education at the priority level of the state. In 1990s and 2000s, educational reform aimed at the improving parity between urban and rural schooling, development of technical and vocation education and training (TVET) higher education and private sector education (PSE). At the same time, curriculum content was made standardized to improve its quality, including rules for enhancing the quality of the teaching staff and for determining the rights and obligations of educational institutions. The government’s more recent of the Double Reduction Policy of 2021 is an attempt at relieving academic pressure and tackling the socioeconomic issues surrounding private tuition.However, the following factors are attained hence a mode of concern; There has been a continued trend in inequalities between the urban and the rural areas, education has not been universal and is still indifferent between the quality it provides to the students now and then, laws remain to be enacted due to China’s orientation of becoming more international and technologically intelligent. Thus, in the course of this paper, these current issues are discussed and the prospects for future development of education law in China are analyzed, focusing on the fact that, while maintaining the proper balance of state regulation, the state’s goals should correspond to the various orientations for the development of education for the population.
LULC Impacts on NDVI and LST: A Case Study on Jashore District, Bangladesh
Authors: Khan Md Shanewaz , Rahman Iftekhar , Tanu Ferdouse Zaman , Ovi Mehedi Hasan , Mahmud Muhammad Sher
Published: January 2025

Studies on land use and land cover (LULC) changes and subsequent effects on environment are not satisfactory in Bangladesh because of the lack of geospatial data and time-series information. By using the open-source Landsat 7 and Landsat 8 imagery data coupled with GIS technology and other ancillary data, the main purpose of this study is to analyze the dynamic changes in LULC in Jashore district of Bangladesh over a 20-year period between 2002 and 2022. Including pre-classification and post-classification identification scenarios, Normalized Difference Vegetation Index (NDVI) analysis was employed to examine the vegetation changes over the period. ArcGIS 10.8 software was employed for analyzing satellite images, and maximum likelihood classification was utilized to create supervised land cover category (water bodies, vegetation, built-up area, and bare soil). Microsoft Excel was used for data analysis and visualization. The findings of this present study indicate notable changes with an increase of 20.77% in urban areas and 14.53% in bare soil. Additionally, there has been a decline of 2.93% in water bodies and 32.37% in vegetation land cover including both natural and anthropogenically modified vegetation such as forests, croplands, grasslands and others. Accuracy evaluations on the land use classification's trustworthiness include Kappa statistics of 0.80 for the year 2022 and 0.65 for the year 2002. A decrease in land surface temperature (LST) in Jashore district over 20 years from 2002 to 2022 has been reported in this study. Although the proportion of vegetation cover has been reduced in 2022, we found a negative correlation between LST and NDVI. Along with LULC, the LST is influenced by many atmospheric and ecological parameters. NDVI is dependent on vegetation canopy type, color and density, which could affect the relationship with LST. The findings of this study provide insightful information to ecologists, environmentalists, urban planners, and lawmakers for developing sustainable land management plans and environmental conservation initiatives.
Assessment of Genetic Variability, Heritability and Genetic Advance for Yield and Related Traits in Pumpkin (Cucurbita moschata Duch ex Poir)
Authors: Neha  , Singh Ajeet , Singh Manish Kumar , Singh Himanshu , Kumar Sunil , Dwivedi Varun , Dwivedi S.V. , Mishra A.C.
Published: January 2025

A study on pumpkin genotypes was conducted at Vegetable Research Farm of Banda University of Agriculture and Technology during the 2022 Kharif season. This study aimed to assess genetic variability, genotype performance, variation coefficients, heritability, and percentage genetic advance for pumpkin genotypes. Genetic variability, heritability, and genetic advance are critical parameters in plant breeding, as they help identify traits with potential for improvement through selection. This experiment was conducted in a Randomized Block Design with three replications to evaluate 18 pumpkin genotypes.  The mean sum of squares attributed to genotypes exhibited significant differences (p < 0.01) across all characters, indicating pronounced genetic variability. Furthermore, phenotypic (PCV) and genotypic (GCV) coefficients of variation were remarkably high for most quantitative traits male/female bud ratio (PCV=37.90% and GCV=37.65%) followed by pericarp/seed ball ratio (PCV=31.39% and GCV= 31.07%) fruit yield (PCV=27.48% and GCV= 26.22%), vine length (PCV=22.14% and GCV=21.85%) and leaf area (PCV=20.73% and GCV=20.31%). The investigation demonstrated high heritability (h²) and genetic advance (GA) for the majority of assessed traits namely male/female bud ratio (98.65%) followed by pericarp/seed ball ratio (98.02%), vine length (97.35%), leaf area (95.95%). The genotypes VRPK-22-04, Kashi Harit and Arka Chandan exhibited superior yield performance. Notably, VRPK-2201 and VRPK-02-07 exhibited early maturity and reasonable yields, rendering them suitable cultivars for the Bundelkhand region.
Topsoil removal for Sphagnum establishment on rewetted agricultural bogs
Authors: Käärmelahti Sannimari A. , Fritz Christian , Quadra Gabrielle R. , Gardoki Maider Erize , Gaudig Greta , Krebs Matthias , Temmink Ralph J. M.
Published: November 2023

Rewetting drained agricultural peatlands aids in restoring their original ecosystem functions, including carbon storage and sustaining unique biodiversity. 30–60 cm of topsoil removal (TSR) before rewetting for Sphagnum establishment is a common practice to reduce nutrient concentrations and greenhouse gas emissions, and increase water conductivity. However, the topsoil is carbon-dense and preservation in situ would be favorable from a climate-mitigation perspective. The effect of reduced TSR on Sphagnum establishment and nutrient dynamics on degraded and rewetted raised bogs remains to be elucidated. We conducted a two-year field experiment under Sphagnum paludiculture management with three TSR depths: no-removal (TSR0), 5–10 cm (TSR5), and 30 cm (TSR30) removal. We tested the effects of TSR on Sphagnum establishment and performance, nutrient dynamics, and hotspot methane emissions. After two years, TSR5 produced similar Sphagnum biomass as TSR30, while vascular plant biomass was highest in TSR0. All capitula nitrogen (N > 12 mg/g) indicated N-saturation. Phosphorus (P) was not limiting (N/P < 30), but a potential potassium (K) limitation was observed in year one (N/K > 3). In TSR0, ammonium concentrations were > 150 µmol/l in year one, but decreased by 80% in year two. P-concentrations remained high (c. 100 µmol/l) at TSR0 and TSR5, and remained low at TSR30. TSR30 and TSR5 reduced hotspot methane emissions relative to TSR0. We conclude that all TSR practices have their own advantages and disadvantages with respect to Sphagnum growth, nutrient availability and vegetation development. While TSR5 may be the most suitable for paludiculture, its applicability for restoration purposes remains to be elucidated. Setting prioritized targets when selecting the optimal TSR with peatland rewetting is pivotal.
Motivations for participation in green crowdfunding: Evidence from the UK
Authors: Adamska-Mieruszewska Joanna , Zientara Piotr , Mrzygłód Urszula , Fornalska Anna
Published: November 2023

This study investigates the sociopsychological mechanisms influencing the inclination to provide financial support for environmentally-oriented crowdfunding initiatives. It considers participation in these campaigns as a form of pro-environmental behaviour (PEB) and applies two theoretical frameworks, namely the theory of planned behaviour (TPB) and the value-belief-norm theory (VBN), to to assess their effectiveness in explaining the intention to support such projects. The results demonstrate that both TPB and VBN theories can elucidate the intention to support green crowdfunding projects. However, TPB displays a stronger explanatory capacity compared to VBN, highlighting its suitability in explaining higher-cost PEB activities, like contributing to green campaigns, as suggested in this study. Nevertheless, it is important to note that, as anticipated by the VBN theory, values, norms and beliefs also contribute to the intention to support such projects.
Structural and optical characterization of beta-gallium oxide
Authors: Muramba Valentine W. , Ali Abdulraoof I. A. , Nel Jacqueline M.
Published: November 2023

Demands for cheaper solar cells have led researchers to less complex, low-temperature, vacuum-free thin-film deposition processes, like spray pyrolysis and sol–gel spin coating. Previous studies of β-Ga2O3 thin-film deposition have used aqueous solutions of gallium nitrate which have strong tendencies to form hydroxide precipitates. This leads to the blockage of spray gun nozzles. To avoid precipitation, tetrahydroxogallate (III) ammonium was used as a novelty precursor in this study for the synthesis of β-Ga2O3. In the spray pyrolysis technique, the precursor was deposited on sapphire substrates at 200 ℃, with a carrier gas pressure of 200 kPa. The same precursor, with added monoethanolamine to enhance viscosity, was used in the spin coating method. A polycrystalline β-Ga2O3 structure was obtained by post-annealing films at 750 ℃ in ambient air. The spin-coated films with thicknesses ranging from 165 to 354 nm exhibited an average crystallite size of 17.78 nm and an optical band gap range between 4.80 eV and 4.95 eV. Films produced by spray pyrolysis had thicknesses ranging between 158 and 255 nm, an average crystallite size of 17.55 nm, and a band gap ranging between 4.69 eV and 4.93 eV. From Raman spectroscopy, the molecular vibrational modes Ag and Bg were detected, featuring three blue shifts and two red shifts. Films showed a UV-blue region originating from oxygen and gallium vacancies in the lattice, an important characteristic for good photodetectors and vital for solar cell passivation. When utilizing β-Ga2O3 as dielectric coating, the refractive index between air and solar cells is reduced, enhancing solar energy absorption. Similar results were obtained for both synthesis techniques confirming the reliability of the methods.
A Review of the Constitutive Modelling of Metals and Alloys in Machining Process
Authors: Bakhshan Hadi , Oñate Eugenio , Carbonell i Puigbó Josep Maria
Published: November 2023

Conventional machining still represents a predominant manufacturing process for the production of metal parts. During the last few decades, extensive research has been conducted to develop predictive models to capture complex material response during the machining process. Understanding the plastic behavior of the metals and alloys during machining operations has a great significance for researchers and engineers in both academia and industry. This paper thoroughly reviews the constitutive material models that have been employed thus far in the conventional machining studies. The aim of the paper is to present all significant constitutive models focusing the discussion on the most frequently used. First, we introduce the phenomenological models that depend on the deformation variables including strain, strain rate and temperature. Several extended versions proposed in the literature of these types of models will be reviewed. The techniques to identify the material constant parameters will also be discussed. Second, the proposed physical-based models, a kind of model that relies on the evolution of internal state variables, including dislocation density and grain size, will be addressed. Following that, novel data-driven based constitutive models are briefly debated to highlight their capabilities in order to be exploited in machining analysis. Finally, a concise overview and perspectives for future research efforts are outlined.
Impact of maternal cardiometabolic status after bariatric surgery on the association between telomere length and adiposity in offspring
Authors: San-Cristobal Rodrigo , de Toro-Martín Juan , Guénard Frédéric , Pérusse Louis , Biron Simon , Marceau Simon , Lafortune Payette Annie , Vohl Marie-Claude
Published: November 2023

The impact of bariatric surgery on metabolic and inflammatory status are reflected in the epigenetic profile and telomere length mediated by the changes in the metabolic status of the patients. This study compared the telomere length of children born before versus after maternal bariatric surgery as a surrogate to test the influence of the mother’s metabolic status on children’s telomere length. DNA methylation telomere length (DNAmTL) was estimated from Methylation-EPIC BeadChip array data from a total of 24 children born before and after maternal bariatric surgery in the greater Quebec City area. DNAmTL was inversely associated with chronological age in children (r = − 0.80, p < 0.001) and significant differences were observed on age-adjusted DNAmTL between children born before versus after the maternal bariatric surgery. The associations found between body mass index and body fat percentage with DNAmTL in children born after the surgery were influenced by maternal triglycerides, TG/HDL-C ratio and TyG index. This study reports the impact of maternal bariatric surgery on offspring telomere length. The influence of maternal metabolic status on the association between telomere length and markers of adiposity in children suggests a putative modulating effect of bariatric surgery on the cardiometabolic risk in offspring.
Thyme extract could overcome diabetes-induced reproductive dysfunction by inhibiting oxidative damage and increasing the expression of insulin receptor substrate and pyruvate kinase in the rat sperm
Authors: Raeeszadeh Mahdieh , Shokrollahi Borhan , Akbari Abolfazl , Masumi Sima , Amiri Ali Akbar
Published: November 2023

Objectives

Oxidative stress and disruption of energy metabolism in the reproductive system, especially sperm, play a significant role in diabetes-related infertility. Zataria multiflora Boissis (ZMB), a medicinal plant containing various bioactive compounds, may have efficacy in treating metabolic diseases and reproductive disorders. Therefore, the aim of this study was to investigate the effects of different doses of ZMB extract on diabetes-induced reproductive dysfunction by assessing oxidative damage and the gene expression of insulin receptor substrate (IRS) and pyruvate kinase (PK) in male rats’ sperm.

Methods

Sixty adult male Wistar rats were randomly divided into six groups; control (C), diabetes (D), and diabetic animals treated with glibenclamide (G, 50 mg/kg) and thyme extract (T100, T200, and T400). Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ) (50 mg/kg). Insulin, glucose, oxidative and pro-inflammatory markers in the serum, and gene expressions of IRS, and PK were measured in the stored sperms in the epididymis. Changes in the process of spermatogenesis were assessed through the histological evaluation of the testis. Gas chromatography–mass spectrometry (GC–MS) was used to analyze the quantity and quality of thyme extract.

Key findings

The study results indicated that body weight, food intake, and sperm parameters significantly improved in a dose-dependent manner in the T200 group compared to the other groups. Additionally, in the same group, pro-inflammatory biomarkers, DNA fragmentation, and MDA levels decreased, while the levels of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes in the stored epididymal sperm significantly improved compared to the other groups. The expression of IRS and PK, along with the mean counts of spermatogenesis cell lines (especially Sertoli cells), significantly increased in the T200 group.

In conclusion, thymol appears to alleviate diabetes-induced reproductive dysfunction by inhibiting oxidative damage, improving the metabolic state, and upregulating the expression of IRS and PK genes in the sperm of male rats.
3 Hours of Intermittent Hypoxia Increases Circulating Glucose Levels in Healthy Adults
Authors: Newhouse Lauren Pearl , Joyner Michael J , Curry Timothy B , Limberg Jacqueline K
Published: November 2023

Background

We have previously shown that the carotid body chemoreceptors play an important role in glucoregulation. When the carotid bodies are experimentally desensitized in humans, counterregulatory responses to hypoglycemia are attenuated. Whether carotid body activation conversely increases circulating glucose levels was previously unexamined. We hypothesize plasma glucose levels would be increased in young healthy adults after carotid body activation via acute intermittent hypoxia.

Methods

Seven (6M/1F, 21–34 years) healthy, non‐obese, non‐diabetic, subjects without sleep apnea completed 2 visits randomized to 3‐hours of intermittent hypoxia or continuous normoxia. Oxygen saturation and ventilation (turbine ventilation flow transducer) were measured continuously. Plasma glucose levels and carotid body chemosensitivity (hypoxic ventilatory response, HVR) were measured before and after the 3‐hour protocol. Intermittent hypoxia consisted of 25‐s exposures to hypoxic gas (0.05 FiO2) separated by 2‐min of room air breathing, resulting in 25‐events per hour in which oxygen saturation was significantly reduced (Normoxia: 97%, Hypoxia: 89%, p<0.01).

Results

No changes in plasma glucose (93 vs 93 mg/dL, p=0.92) or HVR (−0.35 vs. −0.39 L/min/%SpO2, p=0.56) were observed after 3‐hours of continuous normoxia. In contrast, circulating glucose levels and chemosensitivity increased after intermittent hypoxia when compared to baseline (92 vs 97 mg/dL, p<0.01) and (−0.39 vs −0.60 L/min/%SpO2, p=0.04), respectively. Additionally, the rise in glucose was significantly correlated with the rise in carotid body chemosensitivity (R=0.38, p=0.05).

Conclusion

Circulating glucose levels increased after 3‐hours of intermittent hypoxia. The significant relationship between HVR and circulating glucose suggests that the carotid bodies may play a role in altered glucose handling. These findings may have important implications for the high prevalence of diabetes in patients with sleep apnea.

Support or Funding Information

DK90541, HL120570
Enhanced skeletal muscle metaboreflex activation in type 2 diabetes
Authors: Mizuno Masaki , Holwerda Seth H , Mitchell Jere H , Smith Scott A , Fadel Paul J
Published: November 2023

The effects of insulin on the central nervous system are becoming increasingly recognized. However, the impact of insulin resistance, as in type 2 diabetes (T2D) patients, on neural cardiovascular control mechanisms remains unclear. Herein, we examined the blood pressure (BP) and sympathetic responses to activation of metabolically sensitive skeletal muscle afferents (i.e., muscle metaboreflex) in T2D patients (post exercise ischemia (PEI) following 40% maximum voluntary contraction isometric handgrip) and in Sprague Dawley rats with low‐dose streptozotocin and high‐fat diet induced T2D (hindlimb intra‐arterial capsaicin administration, 1 μg/100 μL). T2D patients exhibited exaggerated BP and muscle sympathetic nerve activity (SNA) responses during PEI compared to age, and bodyweight matched healthy controls (muscle SNA: T2D, Δ31.7 ± 4.9 vs. Control, Δ16.7 ± 3.6 burst/100 heartbeats, P=0.04). Likewise, in T2D rats, BP and renal SNA responses to capsaicin infusion were augmented compared to normal Sprague Dawley rats (renal SNA: T2D, 432 ± 133 vs. Control, 83 ± 25 units, P=0.003). In the T2D patient study, the greater pressor and muscle SNA response to PEI appeared to be related to insulin resistance (HOMA IR: mean BP, R=0.42, P=0.07; muscle SNA, r=0.602, P=0.006). To better understand the impact of these results, intra‐cerebroventricular (ICV) insulin injection was performed in Sprague Dawley rats. ICV insulin did not affect the pressor responses to muscle contraction. Collectively, these preliminary findings suggest that muscle metaboreflex activation is augmented in T2D and changes in insulin in the central nervous system may not be a primary mechanism.

Support or Funding Information

Supported by NIH‐088422 (S.A.S) and AHA‐20160072 (P.J.F.)
VO2max is Restored after Prolonged Exposure to Moderate Altitude
Authors: Heinicke Katja , Heinicke Ilmar , Jelkmann Wolfgang , Rawlings Pablo , Behn Claus , Gassmann Max
Published: November 2023

The objective of this study was to investigate if prolonged training at moderate altitude leads to complete acclimatization of maximal oxygen uptake (VO2max), and if physical activity at this altitude promotes additional skeletal muscle adaption. VO2max and biochemical analysis in biopsies of the vastus lateralis muscle were determined in nine moderately trained endurance athletes (A) and six inactive control subjects (C) during 6 weeks altitude training at 2800 m. VO2max was decreased after 1 day of altitude exposure compared with baseline values at 670 m in A (44.4 ± 4.2 ml·kg−1·min−1 vs. 50.1 ± 4.1, p ≤ 0.01) and C (38.7 ± 5.0 ml·kg−1·min−1 vs. 42.1 ± 4.6, p ≤ 0.05), and was completely restored after 44 days at altitude in both groups (A, 50.0 ± 5.7 ml·kg−1·min−1; C, 42.8 ± 5.1). Upon return from altitude, VO2max increased as compared to the baseline values in A (55.4 ± 6.3 ml·kg−1·min−1, p ≤ 0.001), and C (44.7 ± 5.4, ml·kg−1·min−1, p ≤ 0.001). Muscle buffer capacity was increased after altitude exposure in both groups with no differences between the groups (A, p ≤ 0.01; C, p = 0.06; altitude vs. baseline values), whereas the activity of the mitochondrial enzymes citrate synthase and 3‐hydroxyl‐CoA‐dehydrogenase remained unchanged. In conclusion, VO2max was restored to baseline values after 6 weeks at 2800 m and muscle buffer capacity was improved in both moderately trained athletes and in inactive controls, suggesting that exercise does not affect the acclimatization process.

Support or Funding Information

This work has been supported by the Eidgenössische Sportkommission (ESK) of the Bundesamtes for Sport (BASPO), Fensterfabrik Albisrieden, Zurich, Switzerland (U. & G. Frei), and the Cortex Biophysik GmbH, Leipzig, Germany.
Mechanically Overloaded Plantaris Muscles Display Ectopic Sarcolipin Expression that is Required for Myofiber Hypertrophy and Remodelling
Authors: Fajardo Val Andrew , Rietze Bradley A , Chambers Paige J , Quadrilatero Joe , Tupling A. Russell
Published: November 2023

Sarcolipin (SLN) is a regulator of the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump and is critical in mediating muscle based thermogenesis and combating diet‐induced obesity. Unpublished work from our laboratory has shown that SLN may be important in maintaining muscle mass. Specifically, in phospholamban overexpressing mice (PlnOE), a mouse model that accurately recapitulates human centronuclear myopathy and exhibits severe muscle atrophy, we found that genetic deletion of Sln exaggerated the loss in soleus muscle size. As this reduction in muscle size was associated with a failure to promote type II fiber hypertrophy and fast‐to‐slow fiber type shifts, we questioned whether Sln deletion would also lead to similar defects in mechanically overloaded plantaris muscles that normally undergo comparable adaptations. To this end, we mechanically overloaded plantaris muscles from wild‐type (WT) and Sln‐null (SlnKO) mice for 2 weeks by performing soleus and gastrocnemius tenotomy. Strikingly, after 2 weeks of adaptation, the plantaris muscles from WT muscles displayed ectopic expression of SLN. This is particularly impressive since SLN's expression is typically restricted to slow‐oxidative muscles from the adult mouse. Furthermore, after two weeks of mechanical overload, the WT plantaris myofibers displayed significant hypertrophy (+22% to +48%, P < 0.0001) and fast‐to‐slow fiber type transitions with a significant increase in type I(P = 0.03) and type IIA (P = 0.05) fiber proportions, respectively; however, these adaptations to mechanical overload were not observed in SlnKO mice. Since calcineurin signaling is largely responsible for the cellular adaptations that occur with the mechanical overload stimulus, we examined the phosphorylation status of its well‐known substrate, nuclear factor of activated T‐cells (pNFAT). Strikingly, WT overloaded plantaris muscles showed significantly lower pNFAT compared with WT sham (−61%, P = 0.01), whereas pNFAT in overloaded SlnKO plantaris muscles was not significantly different from SlnKO sham (P = 0.11). Taken together, these results show for the first time, that SLN is recruited in mechanically overloaded plantaris muscles, and is critical in stimulating calcineurin‐mediated muscle growth and remodeling.

Support or Funding Information

This work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).
Deficits in Excitation‐Contraction Coupling in Aged Mice
Authors: Li Harry , Mázala Davi AG , Chin Eva R
Published: November 2023

Age‐related muscle weakness is due to muscle atrophy as well as decreases in specific tension. This is due, at least in part, to altered innervation and to changes in muscle fibre type. The purpose of this study was to assess changes in excitation‐contraction (e‐c) coupling and fibre‐type specific gene expression in muscle from: i) 18 and 30 mos. old (18MO, 30MO) compared to 6 mos. old (6MO) mice; and ii) young mice with denervation atrophy in a mouse model of spinal muscular atrophy (SMA) vs. wild‐type (WT) control. Single muscle fibre were obtained from the flexor digitorum (FDB) muscle and intracellular Ca2+ transients evaluated using Fura‐2 in response to electrical stimulation. Fibre‐type specific genes expression was assessed by RT‐PCR in tibialis anterior (TA) muscle. FDB resting Fura‐2 ratio was not different in 18MO and 30MO fibres (0.396±0.007, 0.407±0.008, 0.414±0.010 at 6MO, 18MO and 30MO, respectively) or in SMA vs. WT fibres. Peak tetanic Fura‐2 ratio were reduced at the highest stimulation frequencies (70 and 100Hz) in 18MO and 30MO compared to 6MO (70Hz: 1.244±0.074, 1.040±0.057, 1.031±0.076 and 100Hz: 1.282±0.075, 1.094±0.062, 1.022±0.078 at 6MO, 18MO and 30MO, respectively; p<0.05) but not in SMA compared to WT. Troponin I slow gene expression was increased (2.1 and 3.7 –fold in 18MO and 30MO, respectively), muscle creatine kinase (MCK) and GAPDH increased and myoglobin decreased in 30MO only. MCK only was increased in SMA vs. WT TA. These data indicate deficits in e‐c coupling by 18 mos. of age with no further impairments at 30 mos. which are associated with a shift to a slow/glycolytic fibre type. These changes are not evident with denervation, suggesting other causes of e‐c coupling failure and fibre type‐specific gene expression.

Support or Funding Information

This work was supported by the University of Maryland, College Park new investigator funds to ERC. DAGM is supported by NIH Pre‐doctoral Institutional Training Grant T32‐AG000268 to J.M. Hagberg.
Regulation of Skeletal Muscle Protein Translation during Energetic Stress
Authors: Wolff Christopher A , Peelor Frederick F , Petrilli Erin B , Casey Richard M , Allison Joseph T , Hamilton Karyn L , Miller Benjamin F
Published: November 2023

Protein translation is an energetically costly process that is globally repressed during energetic stress, as AMPK is activated and mTOR is inhibited. However, our laboratory has demonstrated that mitochondrial protein translation is maintained or increased during the energetic stress signaling associated with multiple models of slowed aging. The mechanisms allowing for the selective translation of mitochondrial proteins during energetic stress are not well characterized in mammalian systems. Thus, the purpose of this experiment was to examine the mechanisms regulating mitochondrial protein translation during energetic stress signaling. To induce energetic stress signaling, we treated C2C12 myotubes with 6 concentrations (10μM, 50μM, 100μM, 500μM, 1mM, or 2mM) of either buformin, phenformin, or metformin to find the lowest effective concentration for AMPK activation and mTOR inhibition. Western blot analyses demonstrated that 100μM buformin, 50μM phenformin, and 2mM metformin had the most potent AMPK activation (2.7, 2.6, and 2.5 fold increases, respectively) accompanied by inhibition of mTOR, as assessed by ribosomal protein S6 phosphorylation (0.8, 0.6, and 0.4 fold reductions, respectively). These concentrations were subsequently utilized for the mechanistic experiments. To assess the mechanisms regulating protein translation during energetic stress signaling C2C12 myotubes were treated for 24 hours with Buformin, Rapamycin, Metformin+Rapamycin, or a vehicle‐control. After treatment, translation was halted using cycloheximide and RNA was extracted from the C2C12 myotubes. RNA was then nuclease treated to create ribosome protected mRNA fragments, which were purified, utilized to prepare the cDNA library, and analyzed on an Illumina NextSEQ 500. Ribosome protected mRNA fragments (RPFs) were aligned against the current mouse genome, and further bioinformatics processing is underway. Through ribosomal profiling we were able to determine which proteins were selectively translated during energetic stress and whether these proteins differed by specific drug treatment. Further, this analysis provides proteins of interest that may contribute to slowing the aging process. It is expected that these data will provide mechanistic insight into why energetic stress signaling and the associated increase in mitochondrial protein synthesis contributes to the slowed aging phenotype.

Support or Funding Information

This project was supported by National Institute on Aging Grant R01‐AG‐042569
Regulation Of Actin Dynamics By WNT‐5A: Implications For Human Airway Smooth Muscle Contraction
Authors: Koopmans Tim , Kumawat Kuldeep , Menzen Mark , Halayko Andrew , Gosens Reinoud
Published: November 2023

An important pathophysiological feature of asthma is airway hyperresponsiveness (AHR), characterized by exaggerated bronchoconstriction in which the airway smooth muscle (ASM) is fundamentally involved. How the ASM in asthmatics differs from that in non‐asthmatics is a current focus for research. We have shown previously that WNT‐5A is increasingly expressed in ASM of mild to moderate asthmatics. WNT proteins have been implicated in the regulation of cytoskeletal filaments and microtubules, thus, we tested the hypothesis that WNT‐5A is a determinant of the contractile response of ASM through modulation of the cytoskeleton.

Using bovine tracheal smooth muscle strips, we found that following pre‐incubation with recombinant WNT‐5A (500 ng/mL, 48 h), maximum isometric tension induced by histamine was increased (Emax = 1,67 fold of control, p < 0.01). We used immortalized human ASM cells to study the underlying mechanisms. In myocytes loaded with the ratiometric calcium indicator, Fura‐2, we found that neither direct exposure to WNT‐5A nor pre‐incubation (48 h) had any effect on calcium flux handling. Moreover, WNT‐5A treatment for 24 h did not yield any detectable change in mRNA expression of the major calcium channels or receptors (IP3R, SERCA2 and RyR1/2/3). However, while direct exposure to WNT‐5A (10–200 ng/mL, 24 h) had no effect on total alpha smooth muscle actin (α‐SMA) protein, it did lead to increased abundance of filamentous actin (labelled with fluorochrome‐conjugated phalloidin). These changes, which were visible after 2 hours, could be completely prevented by treatment with the Rho kinase inhibitor Y27632 (1 uM). Interestingly, ASM cells serum‐deprived for 7 days to adopt a contractile phenotype also showed greater WNT‐5A protein expression (1,99 fold of control, p < 0.05) compared to the proliferative phenotype (serum‐fed cells). We next assessed whether TGF‐β1 played an upstream role in the WNT‐5A response. We found that WNT‐5A siRNA not only blocked TGF‐β1‐induced actin polymerization, but could also abrogate TGF‐β1‐induced accumulation of α‐SMA protein. In line with this contention, adding WNT‐5A on top of TGF‐β1 synergistically induced α‐SMA protein abundance (2,21 fold of TGF‐β, p < 0.01).

In summary, WNT‐5A promotes increased agonist‐induced airway smooth muscle contraction, most likely by increasing actin polymerization and actin expression in concerted action with TGF‐β1. These results may be relevant in the understanding of AHR and warrant further investigation.

Support or Funding Information

This work was financially supported by a NWO Vidi grant, 016.126.307.
Sodium Nitrate Supplementation Does Not Alter Mitochondrial Bioenergetics In The Heart
Authors: Monaco Cynthia M.F. , Miotto Paula M. , Holloway Graham P.
Published: November 2023

Sodium nitrate has been shown to protect against the development of hypertension mediated heart failure and improve the functional outcomes following doxorubicin‐induced cardiotoxicity. While oral nitrate has several beneficial effects on cardiovascular performance, the mechanism(s) underlying these responses remain poorly defined. In skeletal muscle, the oral consumption of nitrate has been shown to have various biological effects that would be advantageous for optimal cardiac performance, including an increase in excitation‐contraction properties and mitochondrial coupling efficiency. Therefore, the objective of this study was to determine whether oral sodium nitrate consumption improved mitochondrial bioenergetics in the hearts of healthy rats. Male Sprague Dawley rats (~ 20 weeks of age) were randomly assigned to a control (n=8) or supplemented group (n=8), which consisted of consuming sodium nitrate in their drinking water (1 g/L) for 7 days. Mitochondrial respiration in permeabilized muscle fibres from the left ventricle showed no changes in leak respiration, maximal substrate‐supported respiration, or ADP sensitivity (apparent Km) with and without 20 mM creatine and 10 mM phosphocreatine/10 mM creatine. In addition, sodium nitrate supplementation did not alter the rates of mitochondrial H2O2 emission in permeabilized cardiac fibres. Therefore, compared to skeletal muscle, these results suggest that short‐term sodium nitrate does not affect mitochondrial bioenergetics in the heart and additional mechanisms must explain the beneficial effects of consumption on cardiac performance.

Support or Funding Information

supported by NSERC, Canada
Elucidating Interactions Between Human Mas‐related G‐protein Coupled Receptors
Authors: Van Remoortel Samuel , Arora Rohit , Labro Alain J. , Snyders Dirk J. , Buckinx Roeland , Timmermans Jean‐Pierre
Published: November 2023

Introduction

G‐protein coupled receptors (GPCR's) are important targets in drug discovery research because of their extensive regulatory effects on cellular functioning and their profound molecular diversity. A decade ago a new GPCR family, referred to as Mas‐related G‐protein coupled receptors (MRGPR), was discovered and since then shown to be involved in nociception and mast cell physiology. MRGPR are thus interesting targets in pain treatment and neuro‐immune communication. Previously, we were able to show the involvement of MRGPR in gastro‐intestinal neuro‐immune communication and observed a marked co‐expression of MRGPRD, MRGPRE and MRGPRF in the murine enteric nervous system (Avula et al. 2011; Avula et al. 2013). Given the well‐known concept of GPCR heteromerization, as well as its importance in functional modulation, we investigated heteromeric properties of the human orthologues of MRGPRD, MRGPRE and MRGPRF.

Material and methods

Heteromeric MRGPR interactions were efficiently screened using a novel luciferase complementation‐based platform. Further validation of interacting partners was performed with Fluorescence Resonance Energy Transfer (FRET). Cells were transiently transfected with eCFP‐ and/or eYFP‐coupled receptors, and FRET efficiency was determined with acceptor photobleaching. For MRGPRD plasmids, receptor functionality was tested according to the known MRGPRD‐mediated inhibition of KCNQ2‐potassium currents.

Results

Transient transfections with fluorescently tagged vector constructs confirmed membrane expression of the vector protein product. Ligand‐induced activation of tagged MRGPRD resulted in reversible inhibition of KCNQ2‐mediated potassium currents, indicating functionality of fluorescently tagged MRGPR. Interaction screening with luciferase complementation provided clear evidence for heteromeric interactions between MRGPRE and MRGPRF. FRET analysis on transiently co‐transfected cells confirmed this MRGPRE‐MRGPRF interaction.

Conclusion

This study provides novel insights on the interactions between human MRGPR. The observed MRGPRE‐MRGPRF interaction contributes to the further elucidation of the function of these orphan receptors, and strengthens the relevance of heteromeric interactions in their receptor biology. Furthermore, we provide a proof‐of‐concept for the applicability of a new complementation‐based screening assay as a high‐throughput screening tool for protein interactions.

Support or Funding Information

This study was financially supported by FWO grant G019314N.
Activation of Nlrp3 inflammasomes in Mouse Hepatic Stellate Cells during Schistosoma J. Infection
Authors: Meng Nan , Xia Min , Li Pin‐Lan , Tang Wang‐Xian
Published: November 2023

Despite many studies, the molecular mechanisms initiating local inflammatory response and liver fibrosis during Schistosoma J. infection remain poorly understood. The present study hypothesized that NOD‐like receptor family, pyrin domain containing 3 (Nlrp3) inflammasomes serve as an intracellular machinery in hepatic stellate cells (HSCs) to turn on the inflammatory response and thereby instigate liver fibrosis during such infection. We first demonstrated that in mice infected with Schistosoma J. for 6 weeks liver exhibited increased IL‐1β level and collagen deposition at the periphery of the eosinophilic granuloma with Schistosoma J. eggs. When mice were administrated caspase‐1 inhibitor YVAD prior to and after Schistosoma J. infection, both increase in IL‐1β level and collagen deposition in liver were substantially attenuated. Using isolated, cultured mouse HSCs, we further explored the mechanism by which soluble egg antigen (SEA) from Schistosoma J. activates Nlrp3 inflammasomes. It was confirmed that SEA induced the formation of Nlrp3 inflammasomes, as shown by fluorescence confocal microscopy and thereby increased caspase‐1 activity and IL‐1β release from HSCs. All these changes related to Nlrp3 inflammasome formation and activation were found to be blocked by YVAD. In addition, the cathepsin B inhibitor, Ca‐074Me or antioxidant N‐acetyl‐L‐cysteine (NAC), but not the potassium channel blocker, glibenclamide (GLY) were also found to inhibit SEA‐induced Nlrp3 inflammasome formation and activation. These results suggest that Schistosoma J. infection may result in local inflammation by activation of Nlrp3 inflammasome in HSCs through its egg antigen stimulation and that activation of Nlrp3 inflammasomes in HSCs is associated with both redox regulation and lysosomal dysfunction, but not related to potassium channel activation.
Purification and characterization of a small copper carrier found in mammalian blood plasma and in the urine of mice and dogs with copper overload
Authors: Tellez Miguel , Alsky Travis , Dalphin Matthew , Flynn Stephen , Munoz Arturo , Ibarra Denise , Truong Helen , Linder Maria C , Lutsenko Svetlana , Weldy Scott
Published: November 2023

We recently discovered a small copper carrier (SCC), which in most mammals is mainly bound to larger proteins and can be released by EDTA. It normally accounts for at least 10% of total plasma copper, and more than half the total in Atp7b−/− mice and some Labrador retrievers with copper overload, where it circulates in the “free” form and appears in the urine. This suggests SCC is an alternative means of excreting copper when biliary excretion is inhibited. To begin to isolate and characterize SCC, blood plasma of human volunteers (permitted by the university IRB), pigs, cows, sheep and Labrador retriever dogs were analyzed for their quantities or free and protein‐bound SCC, using 10 and 3kDa ultrafiltration and size exclusion chromatography (SEC) with Superdex 200 and peptide FPLC. By MALDI‐TOF, the main component identified as potentially SCC consistently had a mass of 1329.5 Da (not including Cu), identical to that reported for urinary SCC from Atp7b−/− mice. It eluted between vitamin B12 and NADH in small pore SEC and not with Cu‐EDTA, which aggregated. Two amino acids were identified; a third was blocked by sulfate or phosphate. However, solution NMR indicated the samples were not pure. Preliminary data suggested phenyl HIC and anion exchange might be useful purification steps. Plasma from the pig had the highest levels of total SCC and more than 10% in the “free form”. So it was chosen to identify the best steps for large scale SCC purification.
Variation in Exercise Capacity and Responses to Training in Inbred Mouse Strains
Authors: Massett Michael P. , Avila Joshua J. , Kim Seung Kyum
Published: November 2023

Improvements in cardiorespiratory fitness can significantly reduce the risk of all‐cause mortality. However, 7 – 45% of individuals show minimal or no improvements in exercise capacity. The aim of this study was to characterize differences in exercise capacity and training responses across mouse strains and estimate the genetic contribution to exercise capacity. Male mice from 25 inbred strains (n = 5–6/strain) performed a graded exercise test before and after 4 weeks of exercise training. Genome wide association mapping was performed using an efficient mixed model association (EMMA) algorithm to identify quantitative trait loci (QTL) for pre‐ and post‐training exercise capacity and the response to training. Exercise capacity was significantly different among strains when expressed as time (range = 21 – 42 min) and work performed (range = 0.4 – 3.9 kg·m). The responses to training were also different among strains. For the change in time, there was an approximately 4.5‐fold difference between the highest (+7.5 min) and lowest (−1.6 min) responding strains. This difference reached 7 fold when defined as change in work (−0.3 vs. 2.1 kg·m). Significant associations (P < 0.05) for exercise time were identified on 16 different chromosomes. Similarly, significant associations were identified on nearly all chromosomes for work performed. For training responses, significant associations were identified on 8 different chromosomes. Significant QTL on Chromosomes 3, 6, and 14 were common to most exercise phenotypes. These results indicate genetic background significantly influences responses to exercise training. Furthermore, the novel QTL identified in the current study provide new targets for investigating the mechanisms underlying variation in exercise capacity and training responses.

Support or Funding Information

Supported by NIH grant HL085918 to MPM