Stress Conditions Research Articles

Development and Validation of Fast and Sensitive RP-HPLC Stability-Indicating Method for Quantification of Piroxicam in Bulk Drug.

A simple, rapid, sensitive, and cost-effective green solvent-assisted reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of piroxicam (PRXM). The chromatographic separation was achieved by using a C-18 (250 × 4.6) mm, 5-μm stationary phase and a mobile phase consisting of methanol and 0.1% ortho-phosphoric acid in water in a ratio of (80:20) v/v at a flow rate of 1ml/min. The detection was carried out at a wavelength of 254nm with a constant injection volume of 10μL throughout the analysis. The calibration curve was observed to be linear over the optimum concentration range of 50-300μgmL-1, with an R2 value of 0.9995. The developed method was validated as per the International Council for Harmonisation (ICH) Q2 (R1) guideline. Various parameters like selectivity/specificity, accuracy/recovery, linearity, precision, detection limit, quantitation limit, robustness and stability of analyte in solution were performed for the method validation. The PRXM was evaluated under stressed conditions, including acidic, basic, oxidative, thermal and photolytic, as per ICH Q1 (R2) guidelines. Significant degradation was observed in acidic and basic degradation conditions. Conversely, the drug substance showed stability when exposed to oxidative, photolytic and thermal degradation conditions.

22 Feeding the early gestating gilt during heat stress: Implications towards fetal viability, growth, and development

Abstract Gestational heat stress (GHS) directly impacts litter development and viability and has long term negative postnatal effects on offspring. However, current data on the interactions between GHS, maternal feed energy partitioning, and fetal development measures in pigs are limited. Therefore, the study objective was to gain a greater understanding of how early GHS influences fetal development and viability and whether maternal feed energy partitioning has a role. Gestating gilts (n = 23) were exposed to thermoneutral (TN; n = 11; 18.5 ± 1.2°C) or heat stress (HS; n = 12; cyclic 26 to 38°C) conditions beginning on d 6.0 ± 0.9 of gestation to 33.0 ± 1.7. The HS treatments resulted in greater (P < 0.05) respiration rates and body temperature and HS gilts grew faster (P < 0.05; 0.5 ± 0.1 kg/d) than TN gilts (0.3 ± 0.1 kg/d) as previously reported. Pregnant gilts were individually housed and limit-fed a standard gestating gilt diet (1.82 kg/d; 4.39 Mcal NE intake/d). On gestation d 33 ± 1.7, gilts were harvested and reproductive tract weight, corpus luteum (CL) counts, and fetal weights and counts were recorded. Additionally, fetal images were taken with a digital camera to assess fetal length, width, crown rump length (CRL), and viability using Image J. Fetuses were considered nonviable if CRL was more than 2 standard deviations less than the mean litter CRL or had irregular anatomical traits. All data were analyzed using PROC GLIMMIX with either pregnant gilt as the experimental unit for litter-based measures or individual fetus as the experimental unit for fetal based measures. Gilt NE intake efficiency [average daily gain (ADG); NE intake) was greater for HS (P = 0.04; 0.11 ± 0.01) when compared with TN gilts (0.07 ± 0.01). No fetal ADG (P = 0.26; 0.12 ± 0.04 g/d) or fetal ADG; gilt NE intake differences (P = 0.26; 0.03 ± 0.01) were detected between treatments. Total fetuses per CL were decreased for HS gilts (P = 0.01; 0.72 ± 0.03 fetuses) when compared with TN gilts (0.85 ± 0.04 fetuses). Viable fetuses per CL were reduced for HS gilts (P = 0.05; 0.69 ± 0.01 fetuses) when compared with TN gilts (0.80 ± 0.04 fetuses). The difference between total CL count and total fetuses per gilt tended to be increased for HS (P = 0.08; 6 ± 1 fetus) versus TN gilts (4 ± 1 fetus). No other fetal differences were observed (P > 0.10). In conclusion, early GHS improved gilt growth performance and NE efficiency, but resulted in decreased fertilization rates and reduced fetal viability and had no impact on fetal growth and development.

102 Circadian patterns of behavior, respiration, rectal temperature, saliva melatonin, cytokine concentrations, and milk immune cells are altered by cooling heat-stressed lactating sows

Abstract Lactating sows are particularly vulnerable to heat stress (HS) due to the increased metabolism from milk production. A greater understanding of HS response could assist in mitigating its detrimental effects on animal production. Two core circadian clock genes were pinpointed in a recent genome study as potential mediators of HS response, indicating the possible alteration of circadian clocks by HS exposure. This study aimed to assess how cooling of lactating sows under natural heat stress conditions in the summer influenced circadian patterns of respiration rate (RR), rectal temperature (RT), behaviors, salivary concentration of cortisol and melatonin, milk cell populations and relative abundance of multiple cytokines in saliva. Lactating sows of mixed parities were divided into two treatment groups: treated with electronic cooling pad (C; n=9) and heat-stressed control (H; n=9). Experiments were conducted during two 48 h periods of increased environmental temperatures from summer heatwaves. During the initial 48 h period, rectal temperature (RT) was monitored every 60 min, rectal temperature (RT) every 30 min, and behaviors of eating, standing, sitting, laying, sleeping, drinking, and nursing every 5 min. Morning milk samples were collected following the completion of the first phase and utilized to categorize and count somatic cells. The second trial was approximately 1 wk later, and during this period saliva samples were collected every 4 h over the 48-h period. Results were analyzed for significant effect of treatments and interaction using periodic regression while circadian patterns were further detected by incorporating a Fourier series model utilizing sine-cosine variables that denote a fit and/or shift in circadian pattern. The analysis revealed that cooling decreased RT and RR while also modifying circadian patterns (P < 0.05). Cooling had no impact on the percentage of time spent in any activity over the 48-h period (P > 0.05). Nevertheless, the daily patterns of eating, standing, and sitting were different between the treatments (P < 0.05). A shift in eating time to later in the day in H sows relative to C sows related to a second rise in RT during the dark phase of the light-dark cycle (P < 0.05). Saliva melatonin concentrations were increased in C sows and circadian pattern altered relative to H (P < 0.05), but there was no effect on salivary cortisol concentrations (P > 0.05). Cooling reduced milk lymphocytes (P < 0.05) and influenced diurnal pattern of saliva cytokines including interleukins (IL) IL-1 alpha, IL-1 beta, IL-10, and IL-15 and transforming growth factor-beta 1. In conclusion, the cooling of heat-stressed sows altered the circadian patterns of physiology, behavior and melatonin, suggesting the need for additional research to investigate whether circadian disruption contributes to reduced production efficiency observed in HS animals.

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Impact of a heterogeneous environment on the population expansion of the harmful plant Iris ruthenica Ker-Gawl. in the high mountain grasslands

Iris ruthenica Ker-Gawl. (Russian iris) is a perennial, clonal, herbaceous plant that is spread across the degraded mountain grasslands in northern Xinjiang. In this study, to explore the breeding system and the impact of slope orientation on the flowering phenology, pollination characteristics, and other aspects of I. ruthenica, we used a combination of field observations and controlled experiments to compare the population density, flowering phenology, floral characteristics, breeding system, and pollinator type and behavior of plants on different slope orientations. Vegetation coverage on the north (N), southwest (SW), and southeast (SE) slopes was 90%, 67%, and 53%, respectively. Differences in plant height and diameter were observed between the SE slope and the N and SW slopes, whereas the N and SW slopes were similar in these respects. The SE slope exhibited the earliest initial flowering time, followed by the SW and N slopes. The diameter of the corolla, distance between the stigma and anther, length of the anther and ovary, number of pollen grains, and number of ovules on the N slope were smaller than those on the SE and SW slopes, whereas those of the SE and SW slopes were similar. Artificial pollination experiments showed that neither bagging nor unbagging resulted in seed formation after emasculation for all slope orientations. The pollinating insects of I. ruthenica included Bombus sp., Amegilla leptocoma, Andrena sp., and Halictus sp.; the types and numbers of pollinating insects differed among slopes. In summary, on SE and SW slopes with high temperatures and sufficient sunlight, this species attracted pollinators and provided them with more opportunities to visit and pollinate by flowering early, with large numbers of blooms, and a longer flowering period and lifespan, ensuring successful reproduction. Under unfavorable conditions, such as insufficient pollinators and limited activity caused by the more stressful environmental conditions of the N slope (including low temperature and insufficient light), this species ensures a certain seed yield by increasing its self-compatibility.

Quantitative proteomics investigating the intrinsic adaptation mechanism of Aeromonas hydrophila to streptomycin.

Aeromonas hydrophila, a prevalent pathogen in the aquaculture industry, poses significant challenges due to its drug-resistant strains. Moreover, residues of antibiotics like streptomycin, extensively employed in aquaculture settings, drive selective bacterial evolution, leading to the progressive development of resistance to this agent. However, the underlying mechanism of its intrinsic adaptation to antibiotics remains elusive. Here, we employed a quantitative proteomics approach to investigate the differences in protein expression between A. hydrophila under streptomycin (SM) stress and nonstress conditions. Notably, bioinformatics analysis unveiled the potential involvement of metal pathways, including metal cluster binding, iron-sulfur cluster binding, and transition metal ion binding, in influencing A. hydrophila's resistance to SM. Furthermore, we evaluated the sensitivity of eight gene deletion strains related to streptomycin and observed the potential roles of petA and AHA_4705 in SM resistance. Collectively, our findings enhance the understanding of A. hydrophila's response behavior to streptomycin stress and shed light on its intrinsic adaptation mechanism.

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

Influence of Cold Stress on Physiological and Phytochemical Characteristics and Secondary Metabolite Accumulation in Microclones of Juglans regia L.

The current study investigated the impact of cold stress on the morphological, physiological, and phytochemical properties of Juglans regia L. (J. regia) using in vitro microclone cultures. The study revealed significant stress-induced changes in the production of secondary antioxidant metabolites. According to gas chromatography-mass spectrometry (GC-MS) analyses, the stress conditions profoundly altered the metabolism of J. regia microclones. Although the overall spectrum of metabolites was reduced, the production of key secondary antioxidant metabolites significantly increased. Notably, there was a sevenfold (7×) increase in juglone concentration. These findings are crucial for advancing walnut metabolomics and enhancing our understanding of plant responses to abiotic stress factors. Additionally, study results aid in identifying the role of individual metabolites in these processes, which is essential for developing strategies to improve plant resilience and tolerance to adverse conditions.

Genome-wide identification and expression analysis of the SET domain-containing gene family in potato (Solanum tuberosum L.)

Genes containing the SET domain can catalyse histone lysine methylation, which in turn has the potential to cause changes to chromatin structure and regulation of the transcription of genes involved in diverse physiological and developmental processes. However, the functions of SET domain-containing (StSET) genes in potato still need to be studied. The objectives of our study can be summarized as in silico analysis to (i) identify StSET genes in the potato genome, (ii) systematically analyse gene structure, chromosomal distribution, gene duplication events, promoter sequences, and protein domains, (iii) perform phylogenetic analyses, (iv) compare the SET domain-containing genes of potato with other plant species with respect to protein domains and orthologous relationships, (v) analyse tissue-specific expression, and (vi) study the expression of StSET genes in response to drought and heat stresses. In this study, we identified 57 StSET genes in the potato genome, and the genes were physically mapped onto eleven chromosomes. The phylogenetic analysis grouped these StSET genes into six clades. We found that tandem duplication through sub-functionalisation has contributed only marginally to the expansion of the StSET gene family. The protein domain TDBD (PFAM ID: PF16135) was detected in StSET genes of potato while it was absent in all other previously studied species. This study described three pollen-specific StSET genes in the potato genome. Expression analysis of four StSET genes under heat and drought in three potato clones revealed that these genes might have non-overlapping roles under different abiotic stress conditions and durations. The present study provides a comprehensive analysis of StSET genes in potatoes, and it serves as a basis for further functional characterisation of StSET genes towards understanding their underpinning biological mechanisms in conferring stress tolerance.

A novel 2D-electrophoresis method for the simultaneous visualization of phosphorylated and O-GlcNAcylated proteoforms of a protein.

Post-translational modifications (PTMs), such as phosphorylation and O-N-acetyl-β-d-glucosaminylation (O-GlcNAcylation), are involved in the fine spatiotemporal regulation of protein functions, and their dynamic interplay is at the heart of protein language. The coexistence of phosphorylation and O-GlcNAcylation on a protein leads to the diversification of proteoforms. It is therefore essential to decipher the phosphorylation/O-GlcNAcylation interplay on protein species that orchestrates cellular processes in a specific physiological or pathophysiological context. However, simultaneous visualization of phosphorylation and O-GlcNAcylation patterns on a protein of interest remains a challenge. To map the proteoforms of a protein, we have developed an easy-to-use two-dimensional electrophoresis method with a single sample processing permitting simultaneous visualization of the phosphorylated and the O-GlcNAcylated forms of the protein of interest. This method, we termed 2D-WGA-Phos-tag-PAGE relies on proteoforms retardation by affinity gel electrophoresis. With this novel approach, we established the cartography of phospho- and glycoforms of αB-crystallin and desmin in the whole extract and the cytoskeleton protein subfraction in skeletal muscle cells. Interestingly, we have shown that the pattern of phosphorylation and O-GlcNAcylation depends of the subcellular subfraction. Moreover, we have also shown that proteotoxic stress condition increased the complexity of the pattern of PTMs on αB-crystallin.

Long-term, medium-term and acute stress response of urban populations of Eurasian red squirrels affected by different levels of human disturbance.

Animals in urban areas often encounter novel and potentially stressful conditions. It is important to understand how wildlife cope with anthropogenic disturbance. To investigate this specific adaptation we live-trapped squirrels in two study sites in Warsaw: a forest reserve and an urban park and we estimated stress responses at three levels: long-term and medium-term stress (the level of stress hormones, i.e. cortisol and cortisone concentrations, in hair and feces) and acute reaction to human-induced stress (measured during handling with the aid of the three indices: breath rate, struggle rate, and vocalization). According to GLMM models no difference in the stress hormones level was found between the two populations. The only differences in cortisol concentrations clearly depended on the season, i.e. being higher in autumn and winter comparying to other seasons. There was no influence of sex, or reproductive status on stress hormones. Forest squirrels had significantly higher breath rates, suggesting they were more stressed by handling. There was no difference in the struggle rate between study areas, this index was mostly affected by season (i.e. being highest in winter). First-trapped squirrels vocalized less than during the subsequent trappings. Assumingly, during the first, and more stressful trapping, squirrels used 'freezing' and/or little vocalization, while during next captures they used alarm calls to warn conspecifics. Overall, we showed that the two squirrel populations differed only in terms of their breath rate. This suggests that they did not differ in medium-term and long-term stress in general, but they can differ in acute response to handling. This also suggests that both populations were similarly affected by environmental factors. The lack of clear effects may also be due to population heterogeneity. Thus, in order to assess the effects of anthropogenic stressors a broader range of indicators and diverse analytical methods, including behavioral analyses, should be employed.

Grandiose narcissism associates with higher cognitive performance under stress through more efficient attention distribution: An eye-tracking study.

Narcissism is a part of the Dark Triad that consists also of the traits of Machiavellianism and psychopathy. Two main types of narcissism exist: grandiose and vulnerable narcissism. Being a Dark Triad trait, narcissism is typically associated with negative outcomes. However, recent research suggests that at least the grandiose type may be linked (directly or indirectly) to positive outcomes including lower levels of psychopathology, higher school grades in adolescents, deeper and more strategic learning in university students and higher cognitive performance in experimental settings. The current pre-registered, quasi-experimental study implemented eye-tracking to assess whether grandiose narcissism indirectly predicts cognitive performance through wider distribution of attention on the Raven's Progressive Matrices task. Fifty-four adults completed measures of the Dark Triad, self-esteem and psychopathology. Eight months to one year later, participants completed the Raven's, while their eye-movements were monitored during high stress conditions. When controlling for previous levels of psychopathology, grandiose narcissism predicted higher Raven's scores indirectly, through increased variability in the number of fixations across trials. These findings suggest that grandiose narcissism predicts higher cognitive performance, at least in experimental settings, and call for further research to understand the implications of this seemingly dark trait for performance across various settings.

A comparison of procedures for evaluating and selecting alfalfa landrace germplasm for tolerance to salinity

AbstractIn arid and semiarid regions, salinity may affect alfalfa (Medicago sativa) productivity and survival due to either cultivation on salt‐affected soils or the use of salinized irrigation water. Exploiting germplasm evolved under salt‐stress conditions offers opportunities for crop tolerance improvement. In the first phase of the current study, four reportedly salt‐tolerant landraces originated from stress‐prone areas of West Asia or North Africa and two reference commercial cultivars underwent three evaluation trials according to different methods, namely, seed germination in saline water, in vitro testing of young plants, and greenhouse evaluation of adult plants in Cone‐tainers. Experimental populations obtained by intercrossing landrace genotypes selected according to each evaluation method were subsequently evaluated under stressful field conditions in two regions featuring different salt stress type, namely, southern Tunisia (irrigation with saline water) and northwestern Argentina (rainfed cropping in saline soil). Landrace ranking for salt tolerance differed somewhat depending on the evaluation method, and the proportion of selected plants per landrace depended accordingly on the method. In each field experiment, there were two evaluation phases, and the second phase corresponded in both cases to harsher conditions. The in vitro evaluation and selection resulted in potentially more useful selected germplasm than the other evaluation methods. The field experiments highlighted the large specific adaptation effects that affected the response of salt‐tolerant germplasm across the two regions.

Drosophila Nhe2 Over-expression Induces Autophagic Cell Death.

Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous fashion. In cancer cells, autophagy is often upregulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via over-expression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase-independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.

Nitrosative stress under microaerobic conditions triggers inositol metabolism in Pseudomonas extremaustralis.

Bacteria are exposed to reactive oxygen and nitrogen species that provoke oxidative and nitrosative stress which can lead to macromolecule damage. Coping with stress conditions involves the adjustment of cellular responses, which helps to address metabolic challenges. In this study, we performed a global transcriptomic analysis of the response of Pseudomonas extremaustralis to nitrosative stress, induced by S-nitrosoglutathione (GSNO), a nitric oxide donor, under microaerobic conditions. The analysis revealed the upregulation of genes associated with inositol catabolism; a compound widely distributed in nature whose metabolism in bacteria has aroused interest. The RNAseq data also showed heightened expression of genes involved in essential cellular processes like transcription, translation, amino acid transport and biosynthesis, as well as in stress resistance including iron-dependent superoxide dismutase, alkyl hydroperoxide reductase, thioredoxin, and glutathione S-transferase in response to GSNO. Furthermore, GSNO exposure differentially affected the expression of genes encoding nitrosylation target proteins, encompassing metalloproteins and proteins with free cysteine and /or tyrosine residues. Notably, genes associated with iron metabolism, such as pyoverdine synthesis and iron transporter genes, showed activation in the presence of GSNO, likely as response to enhanced protein turnover. Physiological assays demonstrated that P. extremaustralis can utilize inositol proficiently under both aerobic and microaerobic conditions, achieving growth comparable to glucose-supplemented cultures. Moreover, supplementing the culture medium with inositol enhances the stress tolerance of P. extremaustralis against combined oxidative-nitrosative stress. Concordant with the heightened expression of pyoverdine genes under nitrosative stress, elevated pyoverdine production was observed when myo-inositol was added to the culture medium. These findings highlight the influence of nitrosative stress on proteins susceptible to nitrosylation and iron metabolism. Furthermore, the activation of myo-inositol catabolism emerges as a protective mechanism against nitrosative stress, shedding light on this pathway in bacterial systems, and holding significance in the adaptation to unfavorable conditions.

CsSHMT3 gene enhances the growth and development in cucumber seedlings under salt stress.

Salt stress is one of the major factors limiting plant growth and productivity. Many studies have shown that serine hydroxymethyltransferase (SHMT) gene play an important role in growth, development and stress response in plants. However, to date, there have been few studies on whether SHMT3 can enhance salt tolerance in plants. Therefore, the effects of overexpression or silencing of CsSHMT3 gene on cucumber seedling growth under salt stress were investigated in this study. The results showed that overexpression of CsSHMT3 gene in cucumber seedlings resulted in a significant increase in chlorophyll content, photosynthetic rate and proline (Pro) content, and antioxidant enzyme activity under salt stress condition; whereas the content of malondialdehyde (MDA), superoxide anion (H2O2), hydrogen peroxide (O2·-) and relative conductivity were significantly decreased when CsSHMT3 gene was overexpressed. However, the content of chlorophyll and Pro, photosynthetic rate, and antioxidant enzyme activity of the silenced CsSHMT3 gene lines under salt stress were significantly reduced, while MDA, H2O2, O2·- content and relative conductivity showed higher level in the silenced CsSHMT3 gene lines. It was further found that the expression of stress-related genes SOD, CAT, SOS1, SOS2, NHX, and HKT was significantly up-regulated by overexpressing CsSHMT3 gene in cucumber seedlings; while stress-related gene expression showed significant decrease in silenced CsSHMT3 gene seedlings under salt stress. This suggests that overexpression of CsSHMT3 gene increased the salt tolerance of cucumber seedlings, while silencing of CsSHMT3 gene decreased the salt tolerance. In conclusion, CsSHMT3 gene might positively regulate salt stress tolerance in cucumber and be involved in regulating antioxidant activity, osmotic adjustment, and photosynthesis under salt stress. KEY MESSAGE: CsSHMT3 gene may positively regulate the expression of osmotic system, photosynthesis, antioxidant system and stress-related genes in cucumber.

Nurses’ experiences during the COVID-19 pandemic: A secondary data analysis examining interdisciplinary and peer communication

BackgroundCommunication with interdisciplinary team members was impacted during the start of the COVID-19 pandemic. Bedside nurses managed communication challenges with interdisciplinary team members while working under extremely stressful conditions created by a new and unknown infectious disease. This study examined data collected from a pre-existing qualitative study to answer the question: What communication challenges occurred among nurses and interdisciplinary team members during the beginning months of the COVID-19 pandemic? MethodsA qualitative secondary analysis of an existing dataset was undertaken. The original data were collected using semi-structured interviews conducted with a purposive sample of bedside nurses (n = 17). ResultsUsing thematic analysis, five themes related to communication among nurses and the interdisciplinary team emerged: 1) acknowledging PPE as a communication barrier, 2) questioning how to perform nursing tasks along with increasing workload, 3) developing innovative ways to deliver safe care, 4) experiencing frustrations about lack of communication with interdisciplinary team members, and 5) experiencing both support and criticism that affect working relationships. ConclusionsChallenges associated with communication during the early days of the COVID-19 pandemic became opportunities to build policies and procedures to support collaboration within the healthcare team. Support is necessary to prepare team members to collaborate efficiently during crisis situations. Developing a strategic plan to improve communication, lessen nurse burden, provide mental health support for nurses, while maintaining safe care are priorities in managing future emergency situations.

Abstract PO4-24-05: Fluid forces and hormone levels during mammary gland development drive changes in breast epithelium that are relevant to the progression of postpartum breast cancer

Abstract Postpartum breast cancer (PPBC), diagnosed in the 5-10 years after childbirth, has an elevated risk of metastasis and death. Poor outcomes are thought to be due to factors involved in mammary gland involution, an important stage of mammary gland development. Involution functions to return the mammary gland to the normal post-lactation state and involves activation of multiple processes such as inflammation, wound healing, and lymphangiogenesis. Previously, these processes have been linked to mechanical forces induced by fluid flowing past cells, known as fluid shear stress (FSS). We and others have shown FSS impacts processes associated with lactation and breast cancer metastasis. Herein, we describe the results of a cell model for mimicking lactation and the cessation of lactation as an investigative tool for probing the mechanisms involved in PPBC development and progression. We identified changing levels of FSS as a potential physiologic biomarker through which pathways associated with the progression of PPBC could be identified. The role of fluid shear stress in the progression of cancer remains relatively unexplored, mostly due to practical research barriers. Since FSS cannot be accurately measured in vivo, most research to date has relied on in vitro modelling. In contrast, the role of the involuting mammary gland on the progression of breast cancer has often been studied using rodent models. As a result, studying the interactions between fluid shear stress and involution poses a novel engineering challenge. To address this problem, we developed a bioreactor cell model to enable cell exposure to fluid flow, mimicking forces experienced during lactation, in presence of lactogenic hormones (dexamethasone, insulin, prolactin). Previous work has induced FSS using parallel plate flow chambers, and induced lactation in mammary epithelial cells grown in vitro using lactogenic hormone treatment. To our knowledge, this is the first time these experimental conditions have been studied in combination. We established three distinct stages of treatment, consistent with the fluid shear stress and hormonal levels expected in (1) lactation, (2) cessation of lactation, and (3) involution. To determine whether the model mimicked expected in vivo conditions in the postpartum mammary gland, we used morphological markers and measured the protein expression of β-casein (CSN2), a milk protein that is an established marker of lactation. Each stage was validated and analyzed using proteomic and genomic sequencing. Generated datasets were further analyzed using unsupervised clustering, combining publicly available datasets with our data. We determined that β-casein levels were highest when protein was collected after the lactation stage, followed by an initial drop-off in β-casein expression when fluid shear stress were lowered during the cessation period. The β-casein levels continued at a constant, low level during the involution period, despite a reintroduction of fluid shear stress. This suggests that breast cancer cells are initially responsive to shear stress stimulation, consistent with what would be expected in the postpartum mammary gland in vivo. Proteomic and genomic datasets generated from cells exposed to FSS demonstrated that multiple pathways associated with metastasis are upregulated, as compared to static controls. When combined with publicly available involution datasets, pathways involved in extracellular matrix remodeling, inflammation, and lymphangiogenesis emerged. Multiple targets identified through this analysis have been previously linked to metastatic activities during breast cancer, suggesting that FSS and involution are relevant physiologic biomarkers in the context of PPBC. Work to validate hormonal and fluid shear stress conditions in combination is ongoing. Future work will use the validated model to test relevant targets identified from analysis of generated datasets. Citation Format: Maya Stibbards-Lyle, Kristina Rinker, Laura Hall, Seleem Badawy, Kathy Zhan. Fluid forces and hormone levels during mammary gland development drive changes in breast epithelium that are relevant to the progression of postpartum breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-05.

Subgrade soil response to rail loading: Instability mechanisms, causative factors, and preventive measures

The demand for more efficient heavy-haul rail networks over soft subgrades poses significant geotechnical challenges and requires a comprehensive understanding of stress conditions as well as the failure potential of subgrade soil under moving wheel loads and increasing rail speeds. Unfavourable stress conditions in the subgrade can result in various types of failures, three of which are identified in this article: (i) excessive plastic settlement, (ii) progressive shear failure, and (iii) subgrade fluidisation (mud pumping). Through a series of advanced testing schemes using cyclic triaxial, hollow cylinder, and an in-house dynamic filtration apparatus, critical stress conditions and soil characteristics prone to subgrade instability are discussed. The results demonstrate that under adverse combinations of loading frequency (f) and cyclic stress ratio (CSR) the continuous application of cyclic loads can lead to an unstable state of soil where excess pore pressure and axial strain increase rapidly. This study also reveals that low to medium plasticity soils (PI < 22) are more vulnerable to subgrade fluidisation, where the rapid internal migration of pore water transforms the upper soil to a fluid-like state with substantial loss in soil stiffness. The layered response of soil through dynamic filtration tests showed larger hydrodynamic forces induced by differential hydraulic gradients in the top layer during cyclic loading causes moisture to move upwards. Various factors that can influence soil instability such as the degree of compaction degree, clay content, soil fabric and stress rotation are also addressed in this paper. Finally, novel solutions for stabilising subgrade such as a vertical drain-composite system and the use of eco-friendly biopolymers are presented.

Dynamic response of He bubbles in Fe9Cr1.5W0.4Si F/M steel under in-situ stepwise nanocompression

Inert-gas bubbles in metals have attracted great attention due to their fundamental role in swelling and degradation of materials. Studies of bubble behavior can provide valuable insights for developing advanced materials. Although extensive research has been conducted on bubble behavior under irradiation or thermal conditions, there is still a lack of investigation under both stress and temperature conditions. In this work, in-situ uniaxial compression tests were performed on helium-bubbled Fe9Cr1.5W0.4Si F/M steel using transmission electron microscopy at 923 K to investigate the responses of bubbles with increasing strain. The results revealed a series of bubble responses, including frequent annihilation, occasional coalescence, and a "forced" coalescence achieved by connecting multiple bubbles under high strain. These responses relied on bubble migration and interaction, influenced by the factors such as the He-V ratio, relative bubble position, and sink effect. Furthermore, the migration mechanism of compressed bubbles at high temperatures is considered to be a combination of surface diffusion induced by thermal gradient and volume diffusion induced by stress gradient. These two mechanisms are expected to compete with each other in different temperature and stress. These findings offer crucial experimental insights into the behavior of bubbles in solids exposed to stress and thermal conditions.