Cold Shock Research Articles

The effect of staged versus rapid immersion on the cardio-respiratory components of the cold-shock response

Drowning is one of the leading causes of accidental death. Sudden cold-water immersion (CWI) leads to the stimulation of cutaneous cold receptors causing the cold-shock response (CSR) which includes a gasp, hyperventilation, and tachycardia for several minutes. Only one study has investigated staged CWI (Hayward and French, 1989, Av Space Env Med, 60, 1163-1165) and found a reduction in minute ventilation (V̇E) and respiratory frequency (fB) occurred when immersed to the waist for 30 s followed by to the shoulders. However, it is unclear whether a staged entry with a longer or shorter period at the waist would elicit a greater or lesser CSR. Thus, the aim of this study is to establish whether a staged immersion with a longer period immersed to the waist would attenuate the initial cardio-respiratory responses associated with CWI, when compared with rapid immersion. Ethical approval was gained from the University of Portsmouth Science Faculty Research Ethics Committee (SHFEC 2020-070). Seven male participants completed two, 5 min immersions that occurred at the same time of day (± 2 h) to avoid circadian variation in internal body temperature: a staged immersion (immersion to the iliac crest for 2 min prior to immersion to the acromion process); a rapid immersion (immersion to the acromion process for 5 min). Participants were lowered into stirred water (15°C) at a rate of 4 s to the waist, 4 s to the shoulder. Inspiratory and expiratory data were collected using a ventilatory turbine module and Douglas bags, respectively, whilst heart rate data was collected using a three-lead ECG. Variables were analysed over the first 30 s of immersion by a one-way ANOVA and a Tukey post hoc test. Effect sizes were calculated using Cohen’s d. Staged immersion to the waist resulted in a significantly lower fB (P < 0.05; d = 1.54) and V ̇E (P < 0.01; d = 1.96) over the first 30 s epoch, when compared to rapid immersion. Staged immersion to the shoulders resulted in a lower ventilatory equivalent of oxygen (P < 0.005; d = 1.95). There was no difference in heart rate between immersions. A staged immersion to the waist resulted in a 36.2% lower V̇E and 29.4% lower fB, potentially due to fewer cutaneous cold receptors being stimulated. This reduces the potential to aspirate water due to a lower ventilatory drive, therefore, could reduce drowning incidence attributed to the CSR.

Including the Ensemble of Unstructured Conformations in the Analysis of Protein's Native State by High-Pressure NMR Spectroscopy.

The analysis of pressure induced changes in the chemical shift of proteins allows statements on structural fluctuations proteins exhibit at ambient pressure. The inherent issue of separating general pressure effects from structural related effects on the pressure dependence of chemical shifts has so far been addressed by considering the characteristics of random coil peptides on increasing pressure. In this work, chemically and pressure denatured states of the cold shock protein B from Bacillus subtilis (BsCspB) have been assigned in 2D 1H-15N HSQC NMR spectra and their dependence on increasing hydrostatic pressure has been evaluated. The pressure denatured polypeptide chain has been used to separate general from structural related effects on 1H and 15N chemical shifts of native BsCspB and the implications on the interpretation of pressure induced changes in the chemical shift regarding the structure of BsCspB are discussed. It has been found that the ensemble of unstructured conformations of BsCspB shows different responses to increasing pressure than random coil peptides do. Thus, the approach used for considering the general effects that arise when hydrostatic pressure increases changes the structural conclusions that are drawn from high pressure NMR spectroscopic experiments that rely on the analysis of chemical shifts.

Analyse des Nativen Zustands von Proteinen durch Hochdruck NMR‐Spektroskopie unter Berücksichtigung des Ensembles Unstrukturierter Konformationen

AbstractDie Analyse druckabhängiger Änderungen der chemischen Verschiebung von Proteinen erlaubt es, Aussagen über strukturelle Fluktuationen zu machen, die diese bereits bei Umgebungsdruck aufweisen. Das Problem, allgemeine Effekte, die mit einer Erhöhung des hydrostatischen Drucks einhergehen, von strukturell bedingten Effekten auf die chemische Verschiebung zu trennen, wurde bisher durch das Einbeziehen der Eigenschaften unstrukturierter Modellpeptide angegangen. In dieser Arbeit wurden chemische Verschiebungen in 2D 1H‐15N‐HSQC Spektren von chemisch als auch druckdenaturierten Zuständen des Kälteschockproteins B aus Bacillus Subtilis (BsCspB) zugeordnet und ihre Abhängigkeit von Änderungen des hydrostatischen Drucks ausgewertet. Die durch hydrostatischen Druck denaturierte Polypeptidkette wurde verwendet, um allgemeine Effekte von strukturbedingten Effekten auf 1H und 15N chemische Verschiebungen zu trennen. Daraus resultierende Interpretationen der druckabhängigen Änderungen der chemischen Verschiebungen werden im strukturellen Kontext diskutiert. Es wurde festgestellt, dass das Ensemble unstrukturierter Konformationen von BsCspB anders auf eine Erhöhung des Drucks reagiert, als es unstrukturierte Modellpeptide tun. Daraus kann geschlussfolgert werden, dass der Ansatz, mit dem man allgemeine Effekte, die durch eine Erhöhung des Drucks hervorgerufen werden, abschätzt, die strukturellen Rückschlüsse ändert, die anhand einer Analyse von chemischen Verschiebungen getroffen werden.

PHYLOGENETIC CONNECTIONS AND COMPARISONS OF THE AMINO ACID SEQUENCES OF SEVERAL PEPTIDE INDUCERS OF MICROBIAL ORIGIN- A FOUNDATION STUDY FOR DEVELOPMENT OF NOVEL BIOPESTICIDES

Peptides of microbial origin stand out as one of theprominent tools to elicit plant immunity. These peptides comprise one of thekey strategies of integrated pest management and are considered as candidatesto develop novel biopesticides. Manyresearch investigations have proved their potential in fending off plantpathogens and were described as sustainable plant protection strategies. Thepresent study was attempted to discover phylogenetic relationships and compareamino acid sequence alignments of various peptide elicitors of microbialorigin. Phylogenetic analysis of 33 microbial peptide elicitors resulted in twoclusters, one cluster contained 19 flagellin sequences, which is furtherdivided into one major (15 peptide sequences) and one (4 peptide sequences)minor subclusters. Further amino acid sequence alignments were carried outbased on the evolutionary relationships among the peptides. The amino acidsequence alignment of flagellin sequences using Clustal W did not present conservativeamino acid sequences except Serine (S), Alanine (A) and Aspartic acid (D).These conserved amino acids (SAxD) that are positioned in the protruding loopmay play a vital role in recognition by plant surface receptors. Alignment ofamino acid sequences of cold shock protein, xylanase, elongation factor andharpin from various sources did not present conservative amino acid sequencesexcept glycine. These investigations lay a theoretical foundation for exploringmany more microbial peptides for inducing plant resistance.

Natural variation of immune epitopes reveals intrabacterial antagonism

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

DNA methylase 1 influences temperature responses and development in the invasive pest Tuta absoluta.

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absolutaMeyrick (Lepidoptera: Gelechiidae)-a destructive invasive pest-can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or -12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi-driven regulation of Tuta absoluta.

Survey of Thai Physicians' Practice in Pediatric Septic Shock.

(1) Background: Sepsis management in children is crucial, especially in emergency services. This study aims to evaluate Thai physicians' knowledge gaps in the emergency management of sepsis in children and to evaluate their adherence to the current sepsis clinical practice guidelines. (2) Methods: This is a cross-sectional survey of Thai physicians' management of septic shock in children. The survey was conducted through online questionnaires from March 2019-April 2019. (3) Results: Of the 366 responders, 362 (98.9%) were completed. Most of the responders were general practitioners (89.2%) and pediatricians (10.8%). The time from positive sepsis screening to being evaluated by physicians within 15 min was reported by 83.9%. The most common choice of fluid resuscitation was normal saline solution (77.3%). The practice of a fluid loading dose (20 mL/kg) consistent with the guidelines was 56.3%. The selection of the first vasoactive agent in warm shock (norepinephrine) and cold shock (epinephrine) according to recommendations in the guidelines was 74.3% and 36.2%, respectively. There was a significant difference between general practitioners and pediatricians in terms of knowledge about initial fluid resuscitation and the optimal vasoactive agent in cold shock (p-value < 0.001). In the multivariate model, factors associated with the guideline-based decision-making of vasoactive agent choice for cold shock were specialist training (pediatrician) and the completion of sepsis management training certification, with adjusted odds ratios (AORs) of 7.81 and 2.96, but working experience greater than ten years was inconsistent with the guideline-based decision-making (AOR 0.14). (4) Conclusions: Thai clinicians were unfamiliar with pediatric sepsis therapy standards, specifically the quantity of early fluid resuscitation and the appropriate vasoactive medications for cold shock. To encourage adherence to the guidelines, we propose a regularly required training course on pediatric sepsis management.

Comparative analysis of folding and unfolding dynamics and free-energy landscapes in homologous cold shock proteins with variable thermal stabilities

Cold shock proteins from the mesophilic organism (BsCSP), the thermophilic organism (BcCSP), and the hyperthermophilic organism (TmCSP) are homologous proteins with similar native structure but quite different thermal stabilities, which makes them promising candidates for investigating the general and specific aspects the folding and unfolding dynamics of proteins and free-energy landscapes. In this study, we employed magnetic tweezers to explore the force-dependent folding and unfolding rates of BsCSP, BcCSP, and TmCSP at forces from several pN to tens of pN. Our results indicate similar force-dependent folding rates for these three proteins, while the unfolding rate of BsCSP, BcCSP, and TmCSP decreases sequentially, consistent with previous biochemical findings. Comparing with TmCSP, BcCSP shows lower but sufficient stability with folding free energy of 8.0 kBT, and its force-dependent unfolding rates exhibit tremendous nonlinear behavior deviated from Bell's model like TmCSP. BsCSP has very low stability with folding free energy of only 3.9 kBT, and its force-dependent unfolding rates at 3–10 pN exhibit almost linear behavior. Comprehensive free-energy landscapes are constructed, revealing common barriers that governs folding and unfolding dynamics for BcCSP and TmCSP but a wide merged barrier for BsCSP. Our findings provide valuable insights into the folding and unfolding mechanisms of these CSPs, shedding light on the relationship between protein structure, stability, and mechanical properties. Published by the American Physical Society 2024

A new method for creating androgenetic diploid fish from tetraploid carp sperm and their genetic composition analysis

The application of cold shock to inhibit the extrusion of the second polar body in fertilized eggs is one of the main methods to induce triploid in diploid fish. In this study, we originally planned to induce hexaploid by this method in tetraploid carp. However, among the surviving offspring, we observed only 50.7% tetraploid and 49.3% diploid individuals, which could potentially result from either androgenetic diploids or gynogenetic diploids. The absence of irradiation and the high proportion of diploids attracted us. However, the lack of sex-specific markers for tetraploid carp, makes it difficult to determine their origin. Fortunately, we also carried out the cross of yellow carp (Cyprinus carpio yellow var., 2n, ♀) × tetraploid carp (♂) and red crucian carp (Carassius auratus red var., 2n, ♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis, diploid offspring have also been found, and whether they have undergone gynogenesis or androgenesis can be judged based on phenotype and genotype. In terms of phenotypes, the key phenotypes of two diploids, such as the whisker length, number of lateral scales, and presence of melanin, are consistent with tetraploid carp. In terms of genotypes, through single-copy nuclear gene cloning and fluorescence in situ hybridization (FISH), it is supported that both diploids are androgenetic offspring. In addition, 14.9% of tetraploid offspring were found in red crucian carp (♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis. By the above-mentioned means of detection, it was found that they were created by inhibiting the extrusion of the second polar body of the fertilized eggs. From this it will be seen that the application of cold shock before the extrusion of the second polar body of fertilized eggs cause not only polyploidy, but also the elimination of both second polar body and female nucleus. Previous studies have found that cold shock can induce lethal androgenetic haploids in diploid fish, but these androgenetic diploid fish produced by tetraploid carp sperm were surviving. We summarize the mechanism behind the above phenomenon, which is mainly related to meiosis occurring at the edge of the cell membrane. Cold shock may disrupt the function of the spindle, resulting in the simultaneous retention or extrusion of female nucleus and second polar body. This study will provide valuable insights into androgenesis, polyploidization, and hybridization in fish genetic breeding.

The effect of temperature shock on enhancing sperm motility: A new insight into molecular and cellular mechanisms through current procedures

Asthenozoospermia, characterized by reduced progressive motility (below 32%), is a leading cause of male infertility. Intrauterine Insemination (IUI) serves as the first-line treatment in sperm-related processes, aiming to increase sperm motility. Although Density Gradient Centrifugation (DGC) and swim-up procedures have become primary treatment options, considered the gold standard for IUI, there is a need to optimize these methods with additional factors to enhance motility, particularly for Asthenozoospermia patients.We hypothesize that a brief cold shock, lasting approximately 15–20 min at 17 °C, followed by incubation and sperm processing, can substantially enhance motility. For the first time, the hypothesis and evaluation of post-exposure incubation following a short-term cold shock, within the temperature range of 17–20 °C, were explored and discussed.This hypothesis appears valid and holds importance for several reasons. Firstly, there is a significant relationship between cold shock and its impact on intracellular components and the molecular organization of the cell membrane. Additionally, this process regulates various physiological cellular processes, including transbilayer distribution, acrosome reaction, and sperm motility. Moreover, lowering the temperature induces a molecular flux, facilitating calcium uptake by sperm. This process significantly impacts the quantity of calcium accessible to the sperm, particularly within the flagellum. Choosing the temperature below 20 °C temperature can lead to the activation of calcium-absorbing protein channels in the shortest possible time. Furthermore, a crucial reaction in IUI is sperm capacitation, which occurs when the protein tyrosine phosphorylation pathway, a key factor, is activated. Alternatively, the cold incubation of sperm reveals an increased level of tyrosine phosphorylation and a higher percentage of induced Acrosome Reaction (AR) compared to sperm incubated at normal temperatures. Hence, when sperm is cooled, it initiates a process called tyrosine phosphorylation, ultimately leading to sperm capacitation. Besides these logical reasons, there is limited empirical and incidental support in two cases that confirmed the hypothesis well.

Cold shocks at range edges.

Cold shocks at range edges.

Review of “cold shock” cases in operation of loop heat pipes and related thermal instabilities

Loop Heat Pipes (LHP) are passive two-phase heat transfer devices, driven by capillary pumps that source energy for circulation of working fluid solely from the heat source. They have a proven track record in spacecraft thermal control as well as terrestrial applications. However, LHP may experience partial or complete wick dryout and thermal instabilities at rapid power-up steps or (and) rapid condenser cooling conditions change due to a rapid inflow of subcooled liquid from the condenser into the evaporator. Such cases do not have very wide coverage in literature and are usually presented as a little nuisance in testing of a particular LHP, for which some design workaround was found. Oftentimes suggested solutions trade performance or other design merits for robustness of operation. This article aims to review such case studies where phenomena similar to the described “cold shock” affected LHP performance. The aforementioned instabilities become even more pronounced in grid-like multievaporator LHP having multiple parallel connections between capillary pumps and compensation chambers, as heat and mass transfer between those elements becomes less definite and can eventually increase the heat leak into compensation chambers. Vulnerability of LHP with distributed network of evaporators to seemingly “safe” transient regimes fosters deeper analysis of the “cold shock” phenomenon. With comprehensive definition to the problem and a list of research questions, technical measures for getting rid of it in perspective LHP designs can be foreseen.

Role of electrocardiometry in hemodynamic assessment of children with shock in multisystem inflammatory syndrome following COVID-19: A single-center prospective observational study

Background: The hemodynamic profile in multisystem inflammatory syndrome in children (MIS-C) has not been well described. Therefore, we conducted the study utilizing electrocardiometry (EC) to assess the hemodynamic characteristics in MIS-C patients presenting with shock. Subjects and Methods: This pilot prospective observational study was conducted in the pediatric intensive care unit of a tertiary care hospital. Children between 2 months and 18 years meeting the WHO criteria for MIS-C presenting with shock during second COVID-19 pandemic were included in the study. All patients underwent measurement of hemodynamic profile with EC for the initial 48 h of enrollment. Based on EC assessment at enrolment, the systemic vascular resistance index (SVRi) of 1000–1600 dyn s/cm5/m2 was regarded as normal. The hemodynamic categorization was defined as vasodilatory shock in EC (VDEC) (SVRi &lt;1000 dyn s/[cm5/m2]) and vasoconstrictive shock in EC (VCEC) (SVRi &gt; 1600 dyn s/[cm5/m2]). Results: Thirty-one children met the WHO case definition of MIS-C during the study period. Sixteen children with shock were enrolled and studied. Clinically, 7 (43.75%) children had cold shock, whereas 9 (56.25%) had warm shock. The measured baseline (mean [standard deviation]) hemodynamic variables were cardiac index (CI) of 6 ± 1.41 L/min/m2, stroke volume variation of 23% ±9.6%, SVRi of 954.75 ± 263.35 dyn s/(cm5/m2), and thoracic fluid content of 51.18 ± 17.26 ml. VDEC was the predominant manifestation (87.5%) based on EC. Conclusions: Vasodilatory shock was the predominant phenotype observed in critically ill children with MIS-C with shock by EC assessment.

Hyperosmotic cold shock mouse melanoma cells encapsulated with doxorubicin for targeted treatment of melanoma.

The primary treatment strategies for melanoma include surgical excision, chemotherapy, and radiotherapy. However, the efficacy of these treatments is often limited by drug resistance, recurrence, and severe side effects. Therefore, we aimed to develop a targeted drug delivery system capable of selectively locating tumor sites to minimize systemic toxicity and enhance therapeutic efficacy. This cell drug delivery system can also deliver chemotherapeutic drugs to the tumor microenvironment. We treated B16F10 cells with hyperosmotic cold shock (HCS) to obtain and characterize HCS cells. We then investigated the anti-tumor effects and immune activation capabilities of these cells and explored their potential as a targeted drug delivery system. HCS cells not only maintained an intact cellular structure and tumor antigens but also exhibited high expression of the homologous melanoma-associated antigen glycoprotein 100. These cells demonstrated an exceptional capacity for loading and releasing doxorubicin, which has chemotherapeutic anti-tumor effects. HCS cells can precisely target the tumor microenvironment to minimize systemic toxicity, inducing an immune response by activating CD3+ and CD4+ T cells. HCS cells are non-carcinogenic, with both cellular and tumor antigens intact; thus, they are suitable drug delivery carriers. Our findings highlight the potential of HCS cells for carrying doxorubicin because of their high drug-loading efficiency, effective tumor-targeting and anti-tumor effects. Therefore, our results will facilitate the development of melanoma treatments that have higher efficacy than those in the literature.

Dynamic protein composition of Saccharomyces cerevisiae ribosomes in response to multiple stress conditions reflects alterations in translation activity

Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and directs the catalytic steps of protein synthesis, ribosomal proteins play a role in the assembly of the subunits and are essential for the proper structure and function of the ribosome. To date researchers identified heterogeneous ribosomes in different developmental and growth stages. We hypothesized that under stress conditions the heterogeneity of the ribosomes may provide means to prepare the cells for quick recovery. Therefore the aim of the study was the identification of heterogeneity of ribosomal proteins within the ribosomes in response to eleven stress conditions in Saccharomyces cerevisiae, by means of a liquid chromatography/high resolution mass spectrometry (LC-HRMS) and translation activity tests. Out of the total of 74 distinct ribosomal proteins identified in the study 14 small ribosomal subunit (RPS) and 8 large ribosomal subunit (RPL) proteins displayed statistically significant differential abundances within the ribosomes under stress. Additionally, significant alterations in the ratios of 7 ribosomal paralog proteins were observed. Accordingly, the translational activity of yeast ribosomes was altered after UV exposure, during sugar starvation, cold shock, high salt, anaerobic conditions, and amino acid starvation.

The effect of common paralytic agents used for fluorescence imaging on redox tone and ATP levels in Caenorhabditis elegans.

One aspect of Caenorhabditis elegans that makes it a highly valuable model organism is the ease of use of in vivo genetic reporters, facilitated by its transparent cuticle and highly tractable genetics. Despite the rapid advancement of these technologies, worms must be paralyzed for most imaging applications, and few investigations have characterized the impacts of common chemical anesthetic methods on the parameters measured, in particular biochemical measurements such as cellular energetics and redox tone. Using two dynamic reporters, QUEEN-2m for relative ATP levels and reduction-oxidation sensitive GFP (roGFP) for redox tone, we assess the impact of commonly used chemical paralytics. We report that no chemical anesthetic is entirely effective at doses required for full paralysis without altering redox tone or ATP levels, and that anesthetic use alters the detected outcome of rotenone exposure on relative ATP levels and redox tone. We also assess the use of cold shock, commonly used in combination with physical restraint methods, and find that cold shock does not alter either ATP levels or redox tone. In addition to informing which paralytics are most appropriate for research in these topics, we highlight the need for tailoring the use of anesthetics to different endpoints and experimental questions. Further, we reinforce the need for developing less disruptive paralytic methods for optimal imaging of dynamic in vivo reporters.

Toxicologic Pathology Forum: Apoptosis/Single Cell Necrosis as a Possible Procedural Effect in Primate Brain Following Ice-Cold Saline Perfusion.

Nonclinical studies of test articles (TAs) in nonhuman primates are often designed to assess both biodistribution and toxicity. For this purpose, studies commonly use intravenous perfusion of ice-cold (2°C-8°C) saline to facilitate measurements of TA-associated nucleic acids and proteins, after which tissues undergo later fixation by immersion for histological processing and microscopic evaluation. Intriguingly, minimal apoptosis/single cell necrosis (A/SCN) of randomly distributed neural cells is evident in the cerebral cortex and less often the hippocampus in animals from all groups, including vehicle-treated controls. Affected cells exhibit end-stage features such as cytoplasmic hypereosinophilia, nuclear condensation or fragmentation, and shape distortions, so their lineage(s) generally cannot be defined; classical apoptotic bodies are exceedingly rare. In addition, A/SCN is not accompanied by glial reactions, leukocyte infiltration/inflammation, or other parenchymal changes. The severity is minimal in controls but may be slightly exacerbated (to mild) by TA that accumulate in neural cells. One plausible hypothesis explaining this A/SCN exacerbation is that cold shock (perhaps complicated by concurrent tissue acidity and hypoxia) drives still-viable but TA-stressed cells to launch a self-directed death program. Taken together, these observations indicate that A/SCN in brain processed by cold saline perfusion with delayed immersion fixation represents a procedural artifact and not a TA-related lesion.

Study on the estradiol degradation gene expression and resistance mechanism of Rhodococcus R-001 under low-temperature stress

Estradiol (E2), an endocrine disruptor, acts by mimicking or interfering with the normal physiological functions of natural hormones within organisms, leading to issues such as endocrine system disruption. Notably, seasonal fluctuations in environmental temperature may influence the degradation speed of estradiol (E2) in the natural environment, intensifying its potential health and ecological risks. Therefore, this study aims to explore how bacteria can degrade E2 under low-temperature conditions, unveiling their resistance mechanisms, with the goal of developing new strategies to mitigate the threat of E2 to health and ecological safety. In this paper, we found that Rhodococcus equi DSSKP-R-001 (R-001) can efficiently degrade E2 at 30 °C and 10 °C. Six genes in R-001 were shown to be involved in E2 degradation by heterologous expression at 30 °C. Among them, 17β-HSD, KstD2, and KstD3, were also involved in E2 degradation at 10 °C; KstD was not previously known to degrade E2. RNA-seq was used to characterize differentially expressed genes (DEGs) to explore the stress response of R-001 to low-temperature environments to elucidate the strain's adaptation mechanism. At the low temperature, R-001 cells changed from a round spherical shape to a long rod or irregular shape with elevated unsaturated fatty acids and were consistent with the corresponding genetic changes. Many differentially expressed genes linked to the cold stress response were observed. R-001 was found to upregulate genes encoding cold shock proteins, fatty acid metabolism proteins, the ABC transport system, DNA damage repair, energy metabolism and transcriptional regulators. In this study, we demonstrated six E2 degradation genes in R-001 and found for the first time that E2 degradation genes have different expression characteristics at 30 °C and 10 °C. Linking R-001 to cold acclimation provides new insights and a mechanistic basis for the simultaneous degradation of E2 under cold stress in Rhodococcus adaptation.

Extension mechanism of fracturing in liquid nitrogen fractured coal rock dominated by phase change expansion pressure

In this paper, the liquid nitrogen (LN2) fracturing test research using LN2 phase expansion instead of N2 pressurization was carried out in response to the neglected high phase expansion rate characteristic of LN2, an anhydrous fracturing medium. Expansion pressure measurements, infrared temperature measurements, DIC full-field strain measurements and acoustic emission (AE) signal measurements were used as measurement means. The phase change gas expansion pressure, specimen end-face field, internal fracture signal and end-face temperature changes during the fracturing process were explored. The experimental results show that there was a significant difference in the performance of LN2 cold shock damaged specimens and undamaged specimens during fracturing. The change rule of LN2 phase change expansion pressure under no peripheral pressure went through linearly increasing first and then stabilizing, the slope of the linear phase was 0.029 MPa/s, the expansion pressure in the stabilizing phase was 1.2 MPa and lasted for 12s, and then the specimen ruptured. The initiation pressure of the gas fractured specimen was 2.2 MPa. The surface strain of the specimen was significantly increased during the stabilization stage of expansion pressure, and the maximum values of transverse strain and longitudinal strain were 0.059% and 0.071%, respectively, which were increased by 43.9% and 73.2% within 12 s. At the same time, the number of AE localization points increased from 12 to 78, and the location of the localization points gradually expanded from the two sides of the horizontal slot to the top of the horizontal slot and the outer wall of the specimen. There was a low-temperature zone with obvious temperature gradient in the extended area and no such change existed in the gas fractured specimens. Therefore, the fracture initiation pressure of the damaged specimen is lower than that of the undamaged specimen, and the cracks are more likely to expand and become continuous during the fracturing process, thus producing complex cracks. The use of LN2 with high phase change expansion rate can replace the high-pressure pumping of N2, which is of great significance for the reduction of the implementation time and cost of the LN2 fracturing process.