ROS-induced moderate autophagy of haemocytes confers resistance of Mercenaria mercenaria to air exposure stress.

Air exposure (AE) is a significant environmental stressor that can lead to desiccation, hypoxia, starvation, and disruption of cellular homeostasis in marine bivalves. Autophagy is a highly conserved catabolic pathway that facilitates the degradation of damaged macromolecules and organelles, thereby supporting cellular stress responses. To date, autophagy-mediated resistance mechanisms to AE stress remain largely elusive in bivalves. In this study, we performed a multi-tool approach to investigate the autophagy-related physiological regulation in hard clams (Mercenaria mercenaria) under different duration of AE (T = 0, 1, 5, 10, 20, 30 days). We observed that autophagy of haemocytes was significantly activated on day 5. However, autophagy activity began to significantly decline from day 10 to day 30. Autophagy was significantly inhibited after antioxidant treatment, indicating that reactive oxygen species (ROS) was an endogenous inducer of autophagy. A significant decline in the survival rate of hard clams was observed after injection of ammonium chloride or carbamazepine during AE stress, suggesting that moderate autophagy was conducive for clam survival under AE stress. We also observed DNA breaks and high levels of apoptosis in haemocytes on day 10. Activation of apoptosis lagged behind autophagy, and the relationship between autophagy and apoptosis might shift from antagonism to synergy with the duration of stress. This study provides novel insights into the stress resistance mechanisms in marine bivalves.

RNA-Seq analysis and WGCNA reveal dynamic molecular responses to air exposure in the hard clam Mercenaria mercenaria.

Intertidal bivalves are constantly exposed to air due to daily and seasonal tidal cycles. The hard clam Mercenaria mercenaria is an economically important bivalve species and often subjected to air exposure for more than 10 days during long-distance transportation. Hard clam exhibits remarkable tolerance to air exposure. In this study, we performed RNA sequencing on hemocytes of M. mercenaria exposed to air for 0, 1, 5, 10, 20 and 30 days. The overall and dynamic molecular responses of hard clams to air exposure were revealed by different transcriptomic analysis strategies. As a result, most cytochrome P450 1A and 3A, and monocarboxylate transporter family members were up-regulated during air exposure. Additionally, the dominant molecular process in response to 5-d, 10-d, 20-d and 30-d air exposure was refolding of misfolded proteins in endoplasmic reticulum, lysosome-mediated degradation of phospholipids, protein metabolism and reorganization of cytoskeleton, and activation of anti-apoptotic process, respectively. Our results facilitated comprehensive understanding of the tolerance mechanisms of intertidal bivalves to air exposure.

BTN2A1-BRAF fusion may be a novel mechanism of resistance to osimertinib in lung adenocarcinoma: a case report.

Background: Non-small cell lung cancer (NSCLC) is one of the most common malignancies in the world. Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) indicated for NSCLC that effectively targets sensitive epidermal growth factor receptor mutation and exon20 T790M. Despite initially impressive outcomes, acquired resistance (AR) develops rapidly, typically within 9-13 months, and the mechanisms of resistance are not fully understood. Over the past years, EGFR-TKI and programmed cell death-ligand 1 (PD-L1) inhibitors have been widely used to treat for patients with advanced lung adenocarcinoma. Case Description: Herein we report a middle-aged female who suffered from lung adenocarcinoma based on the pathological diagnosis. Epidermal growth factor receptor exon 19 deletion was detected by next-generation sequencing (NGS). After the patient underwent a series of treatments, including osimertinib, BTN2A1-BRAF fusion was identified. After assessing PD-L1 expression by immunohistochemistry (IHC), the patient was switched to duvalizumab, a PD-L1 inhibitor, but no significant improvements were observed. NGS and IHC assays were conducted to analyze the biopsy and blood samples obtained during treatment. Conclusions: This case substantiates that the acquisition of BTN2A1-BRAF fusion potentially serves as a mechanism of AR to osimertinib in NSCLC. Patients with sensitive epidermal growth factor receptor mutation derive minimal benefit from PD-L1 inhibitors irrespective of the degree of PD-L1 expression in the tumor tissue in IHC. Our case provides a new train of thought for treating this patient population.

Molecular evidence for the adaptive evolution in euryhaline bivalves.

Marine bivalves inhabiting intertidal and estuarine areas are frequently exposed to salinity stress due to persistent rainfall and drought. Through prolonged adaptive evolution, numerous bivalves have developed eurysalinity, which are capable of tolerating a wide range of salinity fluctuations through the sophisticated regulation of physiological metabolism. Current research has predominantly focused on investigating the physiological responses of bivalves to salinity stress, leaving a significant gap in our understanding of the adaptive evolutionary characteristics in euryhaline bivalves. Here, comparative genomics analyses were performed in two groups of bivalve species, including 7 euryhaline species and 5 stenohaline species. We identified 24 significantly expanded gene families and 659 positively selected genes in euryhaline bivalves. A significant co-expansion of solute carrier family 23 (SLC23) facilitates the transmembrane transport of ascorbic acids in euryhaline bivalves. Positive selection of antioxidant genes, such as GST and TXNRD, augments the capacity of active oxygen species (ROS) scavenging under salinity stress. Additionally, we found that the positively selected genes were significantly enriched in KEGG pathways associated with carbohydrates, lipids and amino acids metabolism (ALDH, ADH, and GLS), as well as GO terms related to transmembrane transport and inorganic anion transport (SLC22, CLCND, and VDCC). Positive selection of MCT might contribute to prevent excessive accumulation of intracellular lactic acids during anaerobic metabolism. Positive selection of PLA2 potentially promote the removal of damaged membranes lipids under salinity stress. Our findings suggest that adaptive evolution has occurred in osmoregulation, ROS scavenging, energy metabolism, and membrane lipids adjustments in euryhaline bivalves. This study enhances our understanding of the molecular mechanisms underlying the remarkable salinity adaption of euryhaline bivalves.

Autophagy contributes to increase the content of intracellular free amino acids in hard clam (Mercenaria mercenaria) during prolonged exposure to hypersaline environments.

Marine bivalves in intertidal zones and land-based seawater ponds are constantly subjected to a wide range of salinity fluctuations due to heavy rainfall, intense drought, and human activities. As osmoconformers, bivalves rely primarily on rapid release or accumulation of free amino acids (FAAs) for osmoregulation. Euryhaline bivalves are capable of withstanding hyposaline and hypersaline environments through regulation of physiology, metabolism, and gene expression. However, current understanding of the molecular mechanisms underlying osmoregulation and salinity adaptation in euryhaline bivalves remains largely limited. In this study, RNA-seq, WGCNA and flow cytometric analysis were performed to investigate the physiological responses of hard clams (Mercenaria mercenaria) to acute short-term hyposalinity (AL) and hypersalinity (AH), and chronic long-term hyposalinity (CL) and hypersalinity (CH) stress. We found that amino acids biosynthesis was significantly inhibited and aminoacyl-tRNA biosynthesis was augmented to decrease intracellular osmolarity during hyposaline exposure. Under CH, numerous autophagy-related genes (ATGs) were highly expressed, and the autophagy activity of gill cells were significantly up-regulated. A significant decrease in total FAAs content was observed in gills after NH4Cl treatment, indicating that autophagy was crucial for osmoregulation in hard clams during prolonged exposure to hypersaline environments. To prevent premature or unnecessary apoptosis, the expression of cathepsin L was inhibited under AL and AH, and inhibitors of apoptosis was augmented under CL and CH. Additionally, neuroendocrine regulation was involved in salinity adaption in hard clams. This study provides novel insights into the physiological responses of euryhaline marine bivalves to hyposaline and hypersaline environments.

Effect of heat and hypoxia stress on mitochondrion and energy metabolism in the gill of hard clam.

Aquatic animals suffer from heat and hypoxia stress more frequently due to global climate change and other anthropogenic activities. Heat and hypoxia stress can significantly affect mitochondrial function and energy metabolism. Here, the response and adaptation characteristics of mitochondria and energy metabolism in the gill of the hard clam Mercenaria mercenaria under heat (35 °C), hypoxia (0.2 mg/L), and heat plus hypoxia stress (35 °C, 0.2 mg/L) after 48 h exposure were investigated. Mitochondrial membrane potentials were depolarized under environmental stress. Mitochondrial fusion, fission and mitophagy played a key role in maintain mitochondrion function. The AMPK subunits showed different expression under environmental stress. Acceleration of enzyme activities (phosphofructokinase, pyruvate kinase and lactic dehydrogenase) and accumulation of anaerobic metabolites in glycolysis and TCA cycle implied that the anaerobic metabolism might play a key role in providing energy. Accumulation of amino acids might help to increase tolerance under heat and heat combined hypoxia stress. In addition, urea cycle played a key role in amino acid metabolism to prevent ammonia/nitrogen toxicity. This study improved our understanding of the mitochondrial and energy metabolism responses of marine bivalves exposed to environmental stress.

Genome-wide analysis of the hard clam mitogen-activated protein kinase kinase gene family and their transcriptional profiles under abiotic stress.

Mitogen-activated protein kinase kinase (MAPKK) was the hub component of the Mitogen-activated protein kinase (MAPK) signaling pathway and played an important role in the cellular response to environmental stress. In this study, we identified five MmMAPKK genes in hard clam Mercenaria mercenaria and found that all MmMAPKK genes contain a conserved protein kinase domain. The MmMAPKK genes derived from dispersed duplication were unevenly distributed in three chromosomes. Although the genome size was highly variable among different bivalve mollusks, the number of MAPKK genes was relatively stable. Phylogenetic analysis showed that bivalve MAPKK was divided into five clades, and amino acid sequences of MAPKK from the same clade consisted of similar conserved motifs. The syntenic analysis demonstrated that MmMAPKKs had the highest number of homologous gene pairs with Cyclina sinensis. MmMAPKKs were ubiquitously expressed in all examined tissues, and all MmMAPKK genes were highly expressed in the ovary. MmMAPKK genes showed stress-specific expression under envirionmental stress. MmMAPKK7 showed an upregulated in heat and heat plus hypoxia stress while MmMAPKK1 showed an upregulated in hypoxic stress groups. Dynamic changes of MmMAPKK7, MmMAPKK6 and MmMAPKK1 in hemocytes were observed in response to air exposure. MmMAPKK4 significantly downregulated after air exposure for five days. MmMAPKK7 and MmMAPKK6 might participate in adaptation to low salinity stress. Our results provided useful information about MAPKK and laid a foundation for further studies on MAPKK evolution in the bivalve.

Metabolic response of Mercenaria mercenaria under heat and hypoxia stress by widely targeted metabolomic approach.

In the context of global climatic changes, marine organisms have been exposed to environmental stressors including heat and hypoxia. This calls for the design of multi-stressors to uncover the impact of oceanic factors on aquatic organisms. So far, little is known about the metabolic response of marine organisms, especially bivalves, to the combined effects of heat and hypoxia. In this study, we employed widely targeted metabolomic analysis to study the metabolic response of gills in hard clam, a heat- and hypoxia-tolerant bivalve. A total of 810 metabolites were identified. Results showed that the heat group (HT) and heat plus hypoxia group (HL) had a higher number of differential metabolites than the hypoxia group (LO). Glycolysis was affected by the heat and heat plus hypoxia stress. Moreover, anaerobic metabolic biomarkers were accumulated marking the onset of anaerobic metabolism. Environmental stresses may affect Tricarboxylic acid (TCA) cycle. Accumulation of carnitine and glycerophospholipid may promote fatty acid ß oxidation and maintain cell membrane stability, respectively. The high content of oxidized lipids (i.e., Leukotriene) in HL and HT groups implied that the organisms were under ROS stress. The significantly differential metabolites of organic osmolytes and vitamins might relieve ROS stress. Moreover, accumulation of thermoprotective osmolytes (monosaccharide, Trimethylamine N-oxide (TMAO)) accumulation was helpful to maintain protein homeostasis. This investigation provided new insights into the adaptation mechanisms of hard clam to heat, hypoxia and combined stress at the metabolite level and highlighted the roles of molecules and protectants.

Metabolomics and biochemical assays reveal the metabolic responses to hypo-salinity stress and osmoregulatory role of cAMP-PKA pathway in Mercenaria mercenaria.

Hypo-salinity events frequently occur in marine ecosystem due to persistent rainfall and freshwater inflow, reducing the cytosol osmolarity and triggering cellular stress responses in aquatic organisms. Euryhaline bivalves have developed sophisticated regulatory mechanisms to adapt to salinity fluctuations over a long period of evolution. In this study, we performed multiple biochemical assays, widely targeted metabolomics, and gene expression analysis to investigate the comprehensive metabolic responses to hypo-salinity stress and osmoregulation mechanisms in hard clam Mercenaria mercenaria, which is a euryhaline bivalve species widely cultured in China. During hypo-salinity stress, increased vacuoles appeared in gill filaments. The Na+ and Cl- concentrations in gills significantly decreased because of the up-regulation of Na+/K+-ATPase (NKA) activity. The cAMP content dramatically decreased at 5 d post hypo-salinity stress. Meanwhile, the gene expression levels of adenylate cyclase, proteinkinase A, and sodium and calcium channel proteins were evidently down-regulated, suggesting that cAMP-PKA pathway was inhibited to prevent ambient inorganic ions from entering the gill cells. Antioxidant metabolites, such as serine and Tyr-containing dipeptides, were significantly up-regulated to resist oxidative stress. Glycerolipid metabolism was strengthened to stabilize membrane structure when hypo-salinity stress was prolonged to 5 days. At 1 d post hypo-salinity stress, an increase in alanine and lactate contents marked the initiation of anaerobic metabolism. Acylcarnitines accumulation indicated that fatty acids ß-oxidation was promoted to provide energy for osmoregulation. The potential biomarkers of hypo-salinity stress were identified in hard clams. This study provides novel insights into the metabolic regulatory mechanisms to hypo-salinity stress in euryhaline bivalves.

L-Type Cav 1.2 Calcium Channel-α-1C Regulates Response to Rituximab in Diffuse Large B-Cell Lymphoma.

PURPOSE: One third of patients with diffuse large B-cell lymphoma (DLBCL) succumb to the disease partly due to rituximab resistance. Rituximab-induced calcium flux is an important inducer of apoptotic cell death, and we investigated the potential role of calcium channels in rituximab resistance. EXPERIMENTAL DESIGN: The distinctive expression of calcium channel members was compared between patients sensitive and resistant to rituximab, cyclophosphamide, vincristine, doxorubicin, prednisone (RCHOP) regimen. The observation was further validated through mechanistic in vitro and in vivo studies using cell lines and patient-derived xenograft mouse models. RESULTS: A significant inverse correlation was observed between CACNA1C expression and RCHOP resistance in two independent DLBCL cohorts, and CACNA1C expression was an independent prognostic factor for RCHOP resistance after adjusting for International Prognostic Index, cell-of-origin classification, and MYC/BCL2 double expression. Loss of CACNA1C expression reduced rituximab-induced apoptosis and tumor shrinkage. We further demonstrated direct interaction of CACNA1C with CD20 and its role in CD20 stabilization. Functional modulators of L-type calcium channel showed expected alteration in rituximab-induced apoptosis and tumor suppression. Furthermore, we demonstrated that CACNA1C expression was directly regulated by miR-363 whose high expression is associated with worse prognosis in DLBCL. CONCLUSIONS: We identified the role of CACNA1C in rituximab resistance, and modulating its expression or activity may alter rituximab sensitivity in DLBCL.

ACE2 overexpression inhibits angiotensin II-induced monocyte chemoattractant protein-1 expression in macrophages.

BACKGROUND: The discovery of angiotensin-converting enzyme 2 (ACE2) has shed light on the potential therapy for cardiovascular disease, owing to its key role in the formation of vasoprotective peptide angiotensin (Ang 1-7) from angiotensin (Ang) II. The aim of this study was to evaluate whether ACE2 overexpression could protect human monocyte cell line (THP-1) macrophages from angiotensin II-induced monocyte chemoattractant protein-1 (MCP-1) formation. METHODS: A truncated form of mouse ACE2 (mACE2) was cloned into adenovirus vector (Ad-ACE2) and transfected into THP-1. We examined expression of MCP-1 by administration of a selected Ang (1-7) antagonist (A779) to show the effect of ACE2 overexpression on MCP-1 level induced by AngII. RESULTS: AngII-induced MCP-1 expression increased obviously at 24 h and at the concentration of 10(-6) M. Transduction of THP-1 with Ad-ACE2 resulted in a viral increase in ACE2 activity. This was associated with a significant attenuation of AngII-induced MCP-1 production by 39.6+/-4.0% in THP-1 (mean+/-SEM, n=3). Moreover, expression of MCP-1 increased by 35.1+/-4.2% in Ad-ACE2 transfected THP-1 after incubation with Ang II and A779 compared to that with AngII alone. Collectively, these results indicated that ACE2 overexpression in the THP-1 attenuates AngII-induced MCP-1 production and that this reduction is likely mediated by increased Ang (1-7) level. CONCLUSIONS: ACE2 overexpression may provide a new therapeutic strategy for atherosclerosis by inhibiting MCP-1 production induced by AngII.

A novel binary flooding system of a biobased surfactant and hydrophobically associating polymer with ultralow interfacial tensions.

The alkali free surfactant-polymer flooding system with ultralow interfacial tension is a challenge in enhanced oil recovery at present. A novel alkali free binary flooding system of a biobased zwitterionic surfactant and hydrophobically associating polymer with ultralow interfacial tension at a low surfactant dosage was studied in this paper.

Long-Term Follow-Up on the Donor Foot After Thumb Reconstruction Using Big Toe Wrap-Around Flap in Two Different Operation Methods.

The function of the donor foot has been affected after using big toe wrap-around flap for thumb reconstruction. A modified operation method has been developed to reduce the adverse effect on the donor foot. The current study compared the long-term effect of the classic and the modified operation methods on the donor foot. Gait analysis was carried out, including how the patient walked, the walking speed and walking distance, and how the patient jumped and ran. Plantar pressure was measured while the patient was standing and moving. A total of 45 patients who received the 2 different operation methods were included. The follow-up time was 4-10 years with a mean of 6.5 years. Various degrees of complications occurred for the 21 patients who received the classic operation method. For these patients, plantar pressure of the donor foot was obviously different comparing with the healthy unaffected foot while the patient was standing or walking. For the 24 patients who received the modified operation method, no obvious complications were observed and the plantar pressure of the donor foot and the healthy unaffected foot was similar while the patient was standing or walking. In conclusion, both the classic and the modified operation methods have affected the function of the donor foot after using the big toe wrap-around flap for thumb reconstruction. However, the donor foot was less affected when the modified operation method was used.

MYC and MXI1 protein expression: potential prognostic significance in women with breast cancer in China.

OBJECTIVE: To investigate the expression levels and the clinical significance of MYC and MXI1 proteins in breast cancer. METHODS: The expression levels of MYC and MXI1 were detected by immunohistochemical assay in 166 cases of breast cancer; the relationships among MYC, MXI1 and the clinicopathological parameters were analyzed by χ2 test. Univariate analysis and Cox's proportional hazards model were used to evaluate the prognostic significance of the 2 proteins. RESULTS: 27.71% of the tumor specimens showed high staining intensity for MYC (high-expression group, HEG-MYC) and 22.89% showed high staining intensity for MXI1 (HEG-MXI1); the expression of 2 proteins was negatively correlated (r = -0.177 p = 0.022). The Kaplan-Meier method for survival analysis showed that patients of the MYC-HEG demonstrated a significantly worse disease-specific survival than those of the MYC-low-expression group (LEG) (χ2 = 11.102, p = 0.001). However, patients of the MXI1-HEG had a significantly better disease-specific survival than those of the MXI1-LEG (χ2 = 7.858, p = 0.005). Both univariate analysis and Cox's proportional hazards model indicated that MYC and MXI1 could be independent prognostic molecular markers. CONCLUSION: MYC-HEG and MXI1-LEG levels are associated with poor prognosis in patients with breast cancer, suggesting that they may be useful molecular markers in breast cancer prognosis prediction.