Protein Levels In Response Research Articles

An i-type lysozyme from a crustacean, Pacifastacus leniusculus, functions as a clot-destabilising enzyme

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots.We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the β-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood.

Understanding the molecular mechanisms of drought tolerance in wild soybean (Glycine soja) through multi-omics-based alternative splicing predictions

The absence of adequate moisture resulting from drought poses a significant threat to both the viability and productivity of soybean cultivation. Genetic variation in common soybeans has been noticeably reduced through continuous breeding, and wild relatives with wider genetic diversity are one of the best tools in the search for new tolerance genes. In this study, we selected 139 genes co-expressed at transcript and protein levels in response to drought in Glycine soja through a multi-omics analysis. Drought stress induced co-expression of transcripts and proteins involved in dopamine synthesis within tyrosine metabolism. Polyphenol oxidase, involved in the dopamine synthesis process, was uniquely identified in both DEGs and DEPs, with its protein abundance increased. Co-expression of 9-lipoxygenase during linoleic acid metabolism was confirmed, along with consistent protein accumulation. The co-expression profiling of transcripts and proteins suggests that they may influence their regulatory feedback loops or unknown regulatory mechanisms. Additionally, we predicted the regulation of alternative splicing (AS) in response to drought. AS was predicted for 139 co-expressed genes, and four drought-tolerance-related gene candidate groups were selected. The expression levels of four genes, FT1, CCR1L, RPL18, and uncharacterized LOC114422617, varied depending on their transcript isoforms under drought stress. The occurrence of AS under drought stress may play a role in eliminating susceptibility genes or inducing tolerance genes to adapt to drought stress. Overall, this study reveals a novel mechanism of drought adaptation in wild soybean by predicting the regulation of metabolic pathways and AS events at the transcriptome and proteome levels and presents potential targets for soybean breeding.

PARP inhibition leads to synthetic lethality with key splicing-factor mutations in myelodysplastic syndromes.

Splicing factors are frequently mutated in patients with myelodysplastic syndromes and acute myeloid leukaemia. Recent studies have revealed convergent molecular defects caused by splicing factor mutations, among which R-loop dysregulation and resultant genome instability are suggested as contributing factors to disease progression. On the other hand, understanding how mutant cells survive upon aberrant R-loop formation and genome instability is essential for developing novel therapeutics. The immunoprecipitation was performed to identify R-loops in association with PARP1/poly-ADP-ribosylation. The western blot, immunofluorescence, and flow cytometry assays were used to test the cell viability, cell cycle arrest, apoptosis, and ATM activation in mutant cells following the treatment of the PARP inhibitor. The Srsf2(P95H) knock-in murine hematopoietic cells and MLL-AF9 transformed leukaemia model were generated to investigate the potential of the PARP inhibitor as a therapy for haematological malignancies. The disease-causing mutations in SRSF2 activate PARP and elevate the overall poly-ADP-ribosylation levels of proteins in response to R-loop dysregulation. In accordance, mutant cells are more vulnerable to the PARP inhibitors in comparison to the wild-type counterpart. Notably, the synthetic lethality was further validated in the Srsf2(P95H) knock-in murine hematopoietic cell and MLL-AF9 leukaemia model. Our findings suggest that mutant cells antagonise the genome threat caused by R-loop disruption by PARP activation, thus making PARP targeting a promising therapeutic strategy for myeloid cancers with mutations in SRSF2.

AHNAK2 Promotes the Progression of Pancreatic Ductal Adenocarcinoma by Maintaining the Stability of c-MET.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and rapidly progresses. The overall response rate of PDAC to current treatment methods is still unsatisfactory. Thus, identifying novel targets and clarifying the underlying mechanisms associated with PDAC progression may potentially offer additional treatment strategies. AHNAK2 is aberrantly expressed in a variety of tumors and exerts pro-tumorigenic effects. However, the biological role of AHNAK2 in PDAC remains poorly understood. The expression of AHNAK2 in PDAC and paired non-tumor tissues was detected by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). Lentivirus knockdown was performed to investigate the impact of AHNAK2 on the biological function of pancreatic cancer cells. The subcutaneous cell-derived xenograft (CDX) model and the KPC spontaneous mouse model with AHNAK2 silencing were used to observe the effects of AHNAK2 on tumor growth and prognosis. The expression of c-MET at protein level in response to HGF treatment was assessed using western blot. Our results demonstrated that AHNAK2 was highly expressed in PDAC clinical samples and associated with poor prognosis. Knockdown of AHNAK2 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells. AHNAK2 knockdown or knockout resulted in tumor growth suppression and prolonged survival in mice with PDAC. In addition, AHNAK2 and c-MET expression levels showed a significant positive correlation at the post-transcriptional level. Mechanistically, AHNAK2 promoted tumor progression by preventing c-MET degradation and persistently activating the HGF/c-MET signaling pathway. Overall, our study revealed that AHNAK2 plays an important role in PDAC progression by modulating the c-MET signaling pathway, and targeting AHNAK2 may be an effective therapeutic strategy for PDAC.

Mitochondrial Biogenesis and Remodeling Protein Response to Bouts of High-load vs High-volume Resistance Training

Introduction: The effects of higher-load (HL; sets of 5 repetitions at 80% of 1-RM) versus higher-volume (HV; sets of 10 repetitions at 60% of 1-RM) resistance training (RT) on various molecular outcomes has proven to be similar between the two paradigms. However, the mitochondrial response to such training paradigms remains understudied. Therefore, the purpose of this study was to interrogate protein level responses of HL vs HV RT in proteins related to mitochondrial biogenesis and remodeling. We hypothesized that HV RT would have a greater effect on mitochondrial markers of biogenesis and remodeling than HL RT. Methods: Fifteen resistance trained males participated in 6 weeks of unilateral RT (single-leg leg press and single-leg leg extension) where each leg was randomly assigned to undergo either the HL or HV RT. Biopsies of the vastus lateralis were collected from both legs at PRE and POST and western blots analysis was used to assess mitochondrial proteins related to biogenesis and remodeling. The data collected were analyzed using two-way within-subject repeated measures ANOVAs. Results: Our results show no statistically significant effect of RT condition on markers of mitochondrial biogenesis and remodeling. However, there was a main effect of time in markers of both mitochondrial biogenesis and remodeling with MFN1 decreasing (p = 0.007, ƞ2 = 0.234) and PINK1 increasing (p = 0.009, ƞ2 = 0.222) from PRE to POST. Conclusion: HL and HV RT paradigms do not differentially affect the mitochondrial markers assessed, and our hypothesis that HV RT would elicit a greater response in markers of mitochondrial biogenesis and remodeling was not supported. However, our data supports the idea that mitochondrial adaptations can be stimulated regardless of the RT paradigm. This discovery could be advantageous for individuals selecting a mode of RT training that is most likely to result in the greatest exercise adherence as well as provide multiple health benefits. Furthermore, research into RT induced mitochondrial adaptations between sexes remains understudied, thus future investigations into these bioenergetic responses is warranted. Funding for assays and participant compensation was provided through discretionary laboratory funds from MR. Additionally, a portion of CG Vann’s effort was funded through the National Institutes of Health (R01AG054840). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mRNA-Binding Protein KSRP Limits the Inflammatory Response of Macrophages.

KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3'-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild-type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes involved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), interleukin (IL)-1β and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. At the protein level, KSRP-/- BMDM displayed higher levels of these cytokines after overnight stimulation. Matching results were observed for primary peritoneal macrophages of KSRP-/- mice. These showed higher IL-6, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes an important negative regulator of cytokine expression in macrophages.

Abstract 1723: Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue in pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically divided into two subtypes: PAX-FOXO1 fusion positive RMS (FP-RMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative RMS (FN-RMS), which is marked by mutations in RAS isoforms and some other genes. Chromatin was one of the earliest identified targets for cancer therapy. Several chromatin remodeling proteins are associated with cancer progression processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Components of chromatin remodeling complexes have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact on myogenesis is not well understood. In this study, we will characterize the function of SMARCA1 in RMS cells and skeletal muscle. We hypothesize that SMARCA1 acts to modulate chromatin accessibility and drive RMS tumorigenic growth. Methods: We employed RNA-seq, ATAC-seq, and CUT&RUN to study the impact of SMARCA1 in RMS cells. Furthermore, phenotypic experiments were performed to determine the influence of SMARCA1 on differentiation and proliferation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Our findings show that SMARCA1 is modestly upregulated during differentiation in normal myogenesis. Upon treatment with trametinib, FP-RMS cell lines with SMARCA1 knockout exhibited a dramatic decrease in cell confluency and viability. Intriguingly, the control FP-RMS cell lines demonstrated increased SMARCA1 protein levels in response to trametinib, which could suggest a resistance mechanism to the treatment. Trametinib also affected SMARCA1 mRNA expression in FN-RMS cells. Additionally, it was discovered that the elimination of SMARCA1 not only resulted in a reduction in cell migration but also led to a failure of these cells to develop spheres, unlike the control cells, where the migration gap healed in the usual manner and the spheres were observed. These findings may align with our hypothesis that SMARCA1 regulates chromatin accessibility, potentially impacting adhesion properties and signaling pathways. Analysis of gene clusters in RMS transcriptomic data indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 is also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells, as determined by co-immunoprecipitation experiments. Conclusions: Our findings suggest that SMARCA1 may influence RMS tumor growth. This study might contribute to a deeper understanding of SMARCA1's role in RMS and support its credentialing as a novel therapeutic avenue. Citation Format: Ashwaq Aljabri, Katie Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Bao Tran, Matthew Geisler, Judith Davie, Marielle Yohe. Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1723.

Abstract 937: Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe

Abstract Translation of precision oncology data into feasible precision therapies for hard-to-cure childhood, adolescent and young adult malignancies remains a significant challenge. Identifying therapeutic targets at the protein and pathway level and demonstrating treatment response in personalized models hold great promise, particularly for combination therapies, but may be considered complex and time consuming. Here, we present the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how proteomics combined with rapid patient-derived models can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. Elevated SHMT2 levels were validated by immunohistochemistry and compared to levels across AYA tumors. Based on the suitability for an innovative therapy trial, we prioritized single-agent sertraline, a commercially available anti-depressant medication that inhibits SHMT2, and confirmed a positive drug response in both chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Retrospective expansion in a murine xenograft enabled metabolic tracing on isolated SETTLE- patient-derived xenograft cells using 13C6-glucose confirming SHMT2 activity and response to in vitro treatment. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial of sertraline was initiated by the patient but stopped after 8 weeks after evidence of progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Combining sertraline with the common antibiotic trimethoprim, resulted in enhanced growth inhibition of SETTLE cells in the larval zebrafish xenografts. Significance: Overall, we demonstrate that proteomics and personalized xenograft models may provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact clinical practice. Citation Format: Georgina D. Barnabas, Tariq A. Bhat, Verena Goebeler, Pascal Leclair, Nadine Azzam, Nicole Melong, Jason N. Berman, Jennifer A. Chan, Donna L. Senger, Seth Parker, Christopher A. Maxwell, Gregor S. Reid, Jonathan Bush, Caron Strahlendorf, Rebecca Deyell, C James Lim, Philipp F. Lange. Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 937.

Diel Cycle Proteomics: Illuminating Molecular Dynamics in Purple Bacteria for Optimized Biotechnological Applications.

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes-whether under continuous light or alternating light and dark cycles-could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms.

Cytolethal Distending Toxin Modulates Cell Differentiation and Elicits Epithelial to Mesenchymal Transition.

The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus-infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture. Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis.

Are there specific molecular adaptations of skeletal muscle to repeated sprint training in hypoxia?

Introduction
 Athletes increasingly engage in repeated sprint training that consists of repeated short all-out effort (< 10 s) interspersed by short recoveries (< 60 s). When performed in hypoxia (repeated sprints in hypoxia, RSH), it may lead to greater training effect than in normoxia (RSN). However, the mechanisms underlying this superior training effect of RSH are unclear. Specifically, the role of muscle metabolic response to RSH is still debated and results are heterogeneous. Clarifying the molecular pathways of skeletal muscle adaptations to RSH may thus provide new insights into the role of hypoxia-induced response to training.
 Methods
 Two groups of healthy young men (randomized) performed three training sessions/week for three weeks. Each training session consisted in six series of six sprints (6 s effort/24 s rest) in either normoxia (RSN, n = 7) or normobaric hypoxia (FiO2 = ~13%, RSH, n = 9). Before and after the training period, vastus lateralis muscle biopsies, a repeated sprint ability (RSA) test and a Wingate test were performed. Metabolic muscle adaptations were studied with proteomics and western blotting.
 Results
 RSN and RSH similarly improved power output (p < 0.05) during the RSA test (RSN: + 7.2 ± 7.7% vs. RSH: + 7.9 ± 6.6%) and the Wingate test (RSN: + 1.3 ± 3.6% vs. RSH: + 4.4 ± 5.0%). Proteomics revealed a decrease in several processes involved in oxidative phosphorylation, confirmed by Western Blot with a reduction (p < 0.05) in complexes I (- 19 ± 30%) and V (- 15 ± 24%) protein levels in response to both RSN and RSH. RSN and RSH increased (p < 0.05) protein levels of the hypoxia inducible factor 1α (HIF-1α, + 111 ± 50%) and vascular endothelial growth factor A (VEGFa, + 91 ± 60%). Protein levels of the glycolytic enzyme hexokinase II increased (+ 119 ± 183%, p < 0.05) after both training types. Only RSH induced increased glucose transporter 4 (GLUT4, + 31 ± 18%, p < 0.05) protein level, suggesting specific glycolytic adaptations in response to hypoxia, supported by proteomics data. This specific adaptation may be triggered through the signaling of S100A protein family as we observed an increased S100A13 protein level (+ 467 ± 353%, p < 0.05) and Akt phosphorylation (+ 21 ± 21%, time x group interaction, p < 0.05) as well as several other S100A proteins in proteomics only after RSH training.
 Discussion/Conclusion
 To conclude, RSH did not exhibit in greater performance improvement compared to RSN. However, it further improved the glycolytic phenotype compared to RSN, possibly through specific S100A13 proteins signaling. Thus, we suggest that the reported superiority of RSH to RSN in the literature may stem from superior glycolytic adaptations triggered through the activation of a specific pathway involving S100A13 protein. The potential role of S100A13 protein in skeletal muscle adaptative responses to exercise is novel and the present results open new research perspectives in this field.

8-Oxoguanine DNA glycosylase protects cells from senescence via the p53-p21 pathway.

Cellular senescence is an important factor leading to pulmonary fibrosis. Deficiency of 8-oxoguanine DNA glycosylase (OGG1) in mice leads to alleviation of bleomycin (BLM)-induced mouse pulmonary fibrosis, and inhibition of the OGG1 enzyme reduces the epithelial mesenchymal transition (EMT) in lung cells. In the present study, we find decreased expression of OGG1 in aged mice and BLM-induced cell senescence. In addition, a decrease in OGG1 expression results in cell senescence, such as increases in the percentage of SA-β-gal-positive cells, and in the p21 and p-H2AX protein levels in response to BLM in lung cells. Furthermore, OGG1 promotes cell transformation in A549 cells in the presence of BLM. We also find that OGG1 siRNA impedes cell cycle progression and inhibits the levels of telomerase reverse transcriptase (TERT) and LaminB1 in BLM-treated lung cells. The increase in OGG1 expression results in the opposite phenomenon. The mRNA levels of senescence-associated secretory phenotype (SASP) components, including IL-1α, IL-1β, IL-6, IL-8, CXCL1/CXCL2, and MMP-3, in the absence of OGG1 are obviously increased in A549 cells treated with BLM. Interestingly, we demonstrate that OGG1 binds to p53 to inhibit the activation of p53 and that silencing of p53 reverses the inhibition of OGG1 on senescence in lung cells. Additionally, the augmented cell senescence is shown in vivo in OGG1-deficient mice. Overall, we provide direct evidence in vivo and in vitro that OGG1 plays an important role in protecting tissue cells against aging associated with the p53 pathway.

Correlation Between C-Reactive Protein (CRP) Level and Chemotherapy Response in Colorectal Cancer Patients Based on Carcinoembryonic Antigen (CEA) Levels in Post Definitive Operative and Adjuvant Chemotherapy Three Series at Prof. Dr. I.G.N.G Ngoerah Denpasar Hospital

Background: Colorectal cancer is the third leading cause of cancer-related deaths worldwide. Surgical therapy is the primary modality for early-stage cancer with curative goals; chemotherapy is the direct option in advanced cancer with palliative goals; radiotherapy is one of the primary therapy modalities for rectal cancer. The markers for chemotherapy response include CRP and CEA. Elevated CRP and CEA levels in CRC patients are associated with poor chemotherapy response. Objective: to determine the relationship between C-reactive protein (CRP) levels and chemotherapy response measured by Carcinoembryonic Antigen (CEA) levels in colorectal cancer patients at Prof. Dr. IGNG Ngoerah Hospital. Method: analytic observational study with a retrospective cohort study design. The sample in this study used the total consecutive sampling method. The analysis was carried out with the help of SPSS, including descriptive statistical tests, chi-square tests, and correlation tests with Spearman-Rho. Results: 58 patients were divided into 2 groups of 29 each for the group with high CRP levels (≥5 mg/dl) and low CRP levels (<5 mg/dl). The male subjects with the most age <60 years and stage III. Most duration of illness < 2 years. The relationship between CRP levels and chemotherapy response was based on CEA, RR 3.2 [95% CI 1.1-9.6; p 0.030], and the value of the positive correlation coefficient with strong strength is r = 0.722. Conclusion: high CRP levels are significantly associated with a strong correlation to worse chemotherapy response in colorectal cancer patients characterized by a high risk of increasing CEA levels 3.7 times after three series of FOLFIRI adjuvant chemotherapy.

Multi-omics reveals largely distinct transcript- and protein-level responses to the environment in an intertidal mussel.

Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel, Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments (common garden, low-intertidal or high-intertidal outplant) that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions.

SUMOylation indirectly suppresses activity of the HIF-1α pathway in intestinal epithelial cells

The hypoxia-inducible factor (HIF) is a master regulator of the cellular transcriptional response to hypoxia. While the oxygen-sensitive regulation of HIF-1α subunit stability via the ubiquitin-proteasome pathway has been well described, less is known about how other oxygen-independent post-translational modifications impact the HIF pathway. SUMOylation, the attachment of SUMO (small ubiquitin-like modifier) proteins to a target protein, regulates the HIF pathway, although the impact of SUMO on HIF activity remains controversial. Here, we examined the effects of SUMOylation on the expression pattern of HIF-1α in response to pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) in intestinal epithelial cells. We evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression and inhibition of SUMOylation using a novel selective inhibitor of the SUMO pathway, TAK-981, on the sensitivity of HIF-1α in Caco-2 intestinal epithelial cells. Our findings demonstrate that treatment with TAK-981 decreases global SUMO-1 and SUMO-2/3 modification and enhances HIF-1α protein levels, whereas SUMO-1 and SUMO-2/3 overexpression results in decreased HIF-1α protein levels in response to DMOG. Reporter assay analysis demonstrates reduced HIF-1α transcriptional activity in cells overexpressing SUMO-1 and SUMO-2/3, whereas pretreatment with TAK-981 increased HIF-1α transcriptional activity in response to DMOG. In addition, HIF-1α nuclear accumulation was decreased in cells overexpressing SUMO-1. Importantly, we showed that HIF-1α is not directly SUMOylated, but that SUMOylation affects HIF-1α stability and activity indirectly. Taken together, our results indicate that SUMOylation indirectly suppresses HIF-1α protein stability, transcriptional activity, and nuclear accumulation in intestinal epithelial cells.

Nipsnap1—A regulatory factor required for long-term maintenance of non-shivering thermogenesis

ObjectiveThe activation of non-shivering thermogenesis (NST) has strong potential to combat obesity and metabolic disease. The activation of NST however is extremely temporal and the mechanisms surrounding how the benefits of NST are sustained once fully activated, remain unexplored. The objective of this study is to investigate the role of 4-Nitrophenylphosphatase Domain and Non-Neuronal SNAP25-Like 1 (Nipsnap1) in NST maintenance, which is a critical regulator identified in this study. MethodsThe expression of Nipsnap1 was profiled by immunoblotting and RT-qPCR. We generated Nipsnap1 knockout mice (N1–KO) and investigated the function of Nipsnap1 in NST maintenance and whole-body metabolism using whole body respirometry analyses. We evaluate the metabolic regulatory role of Nipsnap1 using cellular and mitochondrial respiration assay. ResultsHere, we show Nipsnap1 as a critical regulator of long-term thermogenic maintenance in brown adipose tissue (BAT). Nipsnap1 localizes to the mitochondrial matrix and increases its transcript and protein levels in response to both chronic cold and β3 adrenergic signaling. We demonstrated that these mice are unable to sustain activated energy expenditure and have significantly lower body temperature in the face of an extended cold challenge. Furthermore, when mice are exposed to the pharmacological β3 agonist CL 316, 243, the N1–KO mice exhibit significant hyperphagia and altered energy balance. Mechanistically, we demonstrate that Nipsnap1 integrates with lipid metabolism and BAT-specific ablation of Nipsnap1 leads to severe defects in beta-oxidation capacity when exposed to a cold environmental challenge. ConclusionOur findings identify Nipsnap1 as a potent regulator of long-term NST maintenance in BAT.

MdGRF11-MdARF19-2 module acts as a positive regulator of drought resistance in apple rootstock

14-3-3 proteins play an important role in the response of plants to drought resistance. In this study, 14-3-3 protein MdGRF11 was cloned from Malus xiaojinensis, and its positive regulation of drought resistance was verified using Orin calli and M. xiaojinensis plants. The transcription factor MdARF19-2 was further screened for interaction with this protein in vitro and in vivo. We also conducted experiments using Orin calli and found that the overexpression of MdARF19-2 decreased the level of reactive oxygen species (ROS) and increased the activity of enzymes that scavenge ROS in plant materials. This indicates that MdARF19-2 is a positive regulator in the drought resistance of plants. The drought tolerance was further improved by the overexpression of both MdGRF11 and MdARF19-2 in the calli. In addition, we examined several genes related to ROS scavenging with auxin response factor binding elements in their promoters and found that their level of expression was regulated by the MdGRF11-MdARF19-2 module. In conclusion, the enhancement of plant drought resistance by MdGRF11 could be owing to its accumulation at the protein level in response to drought, which then combined with MdARF19-2, affecting the expression of MdARF19-2 downstream genes. Thus, it scavenges ROS, which ultimately improves the resistance of plant to drought stress.

Interfering small ubiquitin modifiers (SUMO) improves the thermotolerance of apple by facilitating the activity of MdDREB2A

Heat stress, which is caused by global warming, threatens crops yield and quality across the world. As a kind of post-translation modification, SUMOylation involves in plants heat stress response with a rapid and wide pattern. Here, we identified small ubiquitin modifiers (SUMO), which affect drought tolerance in apple, also participated in thermotolerance. Six isoforms of SUMOs located on six chromosomes in apple genome, and all the SUMOs were up-regulated in response to heat stress condition. The MdSUMO2 RNAi transgenic apple plants exhibited higher survival rate, lower ion leakage, higher catalase (CAT) activity, and Malondialdehyde (MDA) content under heat stress. MdDREB2A, the substrate of MdSUMO2 in apple, was accumulated in MdSUMO2 RNAi transgenic plants than the wild type GL-3 at the protein level in response to heat stress treatment. Further, the inhibited SUMOylation level of MdDREB2A in MdSUMO2 RNAi plants might repress its ubiquitination, too. The accumulated MdDREB2A in MdSUMO2 RNAi plants further induced heat-responsive genes expression to strengthen plants thermotolerance, including MdHSFA3, MdHSP26.5, MdHSP18.2, MdHSP70, MdCYP18-1 and MdTLP1. In summary, these findings illustrate that interfering small ubiquitin modifiers (SUMO) in apple improves plants thermotolerance, partly by facilitating the stability and activity of MdDREB2A.

Surviving without oxygen involves major tissue specific changes in the proteome of crucian carp (Carassius carassius).

The crucian carp (Carassius carassius) can survive complete oxygen depletion (anoxia) for several months at low temperatures, making it an excellent model for studying molecular adaptations to anoxia. Still, little is known about how its global proteome responds to anoxia and reoxygenation. By applying mass spectrometry-based proteome analyses on brain, heart and liver tissue from crucian carp exposed to normoxia, five days anoxia, and reoxygenation, we found major changes in particularly cardiac and hepatic protein levels in response to anoxia and reoxygenation. These included tissue-specific differences in mitochondrial proteins involved in aerobic respiration and mitochondrial membrane integrity. Enzymes in the electron transport system (ETS) decreased in heart and increased massively in liver during anoxia and reoxygenation but did not change in the brain. Importantly, the data support a special role for the liver in succinate handling upon reoxygenation, as suggested by a drastic increase of components of the ETS and uncoupling protein 2, which could allow for succinate metabolism without excessive formation of reactive oxygen species (ROS). Also during reoxygenation, the levels of proteins involved in the cristae junction organization of the mitochondria changed in the heart, possibly functioning to suppress ROS formation. Furthermore, proteins involved in immune (complement) system activation changed in the anoxic heart compared to normoxic controls. The results emphasize that responses to anoxia are highly tissue-specific and related to organ function.

Proteomic Profiling of Host Response in the Cereal Crop Triticum aestivum to the Mycotoxin, 15-Acetyldeoxynivalenol, Produced by the Fungal Pathogen, Fusarium graminearum.

Deoxynivalenol (DON) is a destructive mycotoxin produced by the fungal pathogen Fusarium graminearum in the devastating cereal disease Fusarium head blight (FHB). Host resistance to FHB has been identified within some of these crops (e.g., wheat, barley, corn); however, identification of how the host reduces the production of, and tolerates, DON to lessen the effects of the disease still requires further discovery. The field of quantitative proteomics is an effective tool for measuring and quantifying host defense responses to external factors, including the presence of pathogens and toxins. Success within this area of research has increased through recent technological developments (e.g., instrument sensitivity) and the accessibility of data analysis programs. One advancement we leverage is the ability to label peptides with isobaric mass tags to allow for sample multiplexing, reducing mass spectrometer run times, and providing accurate quantification. In this protocol, we exemplify this methodology to identify protein-level responses to DON within both FHB-resistant and FHB-susceptible Triticum aestivum cultivars using tandem mass tags for quantitative labeling combined with liquid-chromatography-MS/MS (LC-MS/MS) analysis. Furthermore, this protocol can be extrapolated for the identification of host responses under various conditions, including infection and environmental fluctuations, to elucidate changes in proteomic profiling in diverse biological contexts.