Wild-type Research Articles

Roles of the EF-Hand Domain of PLCZ1 in Establishing Species-Specific Ca 2+ Oscillations in Mouse and Rat Fertilization†.

Periodic increases in cytosolic calcium concentration (Ca2+ oscillations) during mammalian fertilization induce all the events collectively known as egg activation. The sperm-specific phospholipase C, PLC zeta 1 (PLCZ1) represents the "sperm factor" vital for initiating the persistent Ca2+ oscillations in mammals. Despite sequence conservation, the Ca2+ oscillation-inducing properties of the enzyme differ vastly among species, and this is particularly salient between mouse and rat PLCZ1, where the activities vary at least one order of magnitude in favor of the former. As previously shown, injecting wild-type (WT) rat Plcz1 mRNA into metaphase II (MII) mouse eggs induced delayed Ca2+ oscillations with low specific activity compared to the homologous mouse Plcz1 mRNA. We, therefore, sought to uncover the factor(s) diversifying these enzymes by swapping functional domains between species, creating chimeric PLCZ1s. When injected into mouse MII eggs, mouse Plcz1 mRNA with the whole- or part of the EF-hand domains swapped with the rat showed a substantial reduction in activity compared to WT. Consistently, the opposite exchange enhanced the rat's enzyme activity. EF-hand domains 1 and 2 seemed to underlie most differences, and mutations of the divergent amino acids within these domains, substitutions for Glu(m-30; r-29) and Gln(m-58; r-57), changed the activity of both species' PLCZ1s in opposite directions. Collectively, our findings support the view that differences in the sequences of EF-hand domains, especially in several of its charged residues, underpin the distinct PLCZ1 activities between these species, revealing the gametes and species' adaptability to optimize the fertilization signal and early development.

Comprehensive Analysis of Class III Peroxidase Genes Revealed PePRX2 Enhanced Lignin Biosynthesis and Drought Tolerance in Phyllostachys edulis.

Class III peroxidase (PRX) is the key enzyme in lignin biosynthesis and critical for maintaining the redox balance in plants to respond to stress. In moso bamboo (Phyllostachys edulis), a globally significant non-timber forestry species, the potential roles of PRX genes remain largely unknown. In this research, a total of 179 PePRXs was identified on a genome-wide scale in moso bamboo. Phylogenic relationship, conserved motifs, gene structure, collinearity, and cis-acting elements were investigated. Analysis of gene expression indicated that PePRXs exhibited tissue-specific expression and different response patterns to hormones and abiotic stresses. Based on the transcriptome data, ten PePRXs with positive correlations between expression levels and lignification degree were screened out. Among them, PePRX2 was selected as a candidate gene according to the co-expression network. Y1H and Dual-Luc assays demonstrated that PeMYB61 could bind to the promoter of PePRX2 and enhance its transcription. The result of in situ hybridization showed that PePRX2 was specifically expressed in the vascular bundle sheath cells of bamboo shoot. As a secreted protein, PePRX2 was located on the cell wall. Overexpression of PePRX2 led to a significant increase in lignin content in transgenic poplar, indicating that PePRX2 could promote lignin polymerization. In comparison to the wild type, the PePRX2-OE poplar lines exhibited increased peroxidase activity and decreased levels of MDA, O2-, and H2O2 under drought stress, indicating enhanced drought resistance. This thorough analysis of the PRX family in moso bamboo provided new insight into the roles of PePRXs in lignin biosynthesis and drought adaptation.

Role of RAS/BRAF and PIK3CA mutations in tissue and plasma for prognostic assessment in metastatic colorectal cancer (mCRC).

281 Background: Mutations in RAS/BRAF ( RAS/BRAFm ) and PIK3CA ( PIK3CAm ) genes have been described as mechanisms of resistance to anti-EGFR agents in mCRC. However, the utility of liquid biopsy in their baseline detection remains unknown. We aim to analyse oncological outcomes according to RAS/BRAF and PIK3CA mutational status in tissue & plasma in mCRC. Methods: We conducted aretrospective study in patients (pts) with mCRC from 2017 to 2024. RAS/BRAF and PIK3CA mutational status assessment was performed at baseline using Idylla (Biocartis) and Cobas PIK3CA kits, respectively. Cox models were used to analyse overall survival (OS) in RAS/BRAFm and PIK3CAm vs wild-type (WT) population. Results: We analysed 404 paired samples (tissue & plasma) from 202p with mCRC. RAS/BRAFm was described in 45% (91p) in tissue and in 43.6% (88p) in plasma. PIK3CA was tested in 84p, with PIK3CAm detection in 19% (16p) in tissue and 11.9% (10p) in plasma. Liver involvement was more frequent in RAS/BRAFm vs WT (80.6% vs 63.9%; p=0.02), and in PIK3CAm vs WT population (94.4% vs 63.6%; p=0.01). Concordance study (tissue & plasma) for RAS/BRAF and PIK3CA are presented (Table). Subjects with concordant results had worse OS in RAS/BRAFm (18.1m vs 51.1m, HR=2.60; p<0.001) regardless of liver involvement, and showed the same trend in PIK3CAm (22.6m vs 28.1m, p=0.81). Patients with discordant results and plasma RAS/BRAFm or plasma PIK3CAm showed worse OS (23.7 m vs 51.1m; p = 0.006 and 16.4 m vs 42.5 m; p=0.18, respectively) and a lower disease control rate with anti-EGFR therapy (p = 0.04 and p=0.42, respectively). PIK3CAm were evenly distributed in RAS/BRAF WT vs mutated tumors (50% vs 51.5%; p=0.92) and had no independent prognostic impact in OS after stratifying by RAS/BRAF mutational status; RAS/BRAF WT (p=0,93) and RAS/BRAFm (p=0,91). Conclusions: Baseline determination of RAS/BRAFm and PIK3CAm by liquid biopsy in mCRC provide valuable prognostic and predictive information, extending its utility beyond tissue-based analysis. Correlation study of RAS/BRAF and PIK3CA mutational status in tissue and plasma in mCRC. RAS/BRAF tissue PIK3CA tissue RAS/BRAF Plasma Mutated n (%) WT n (%) Total n (%) PIK3CA Plasma Mutated n (%) WT n (%) Total n (%) Mutated n (%) 72 (80) 15 (13.4) 87 (43.1) Mutated n (%) 7 (43.8) 2 (2.9) 9 (10.7) WT n (%) 18 (20) 97 (86.6) 115 (56.9) WT n (%) 9 (56.2) 66 (97.1) 75 (89.3) Total n (%) 90 (100) 112 (100) 202 (100) Total n (%) 16 (100) 68 (100) 84 (100) Positive agreement: 72/90: 80% Positive agreement: 7/16: 43.8% Negative agreement: 97/112: 86.6% Negative agreement: 66/68: 97.1% Overall agreement: 169/202: 83.6% Overall agreement: 73/84: 86.9% KI*: 0.67 KI: 0.49 KI: Kappa Index.

Abstract TMP120: CYB5R3 T117S Enlarges Cerebral Infarct Volume and Platelet Activation in Mice Subjected to Transient MCAO-induced Ischemic Stroke

Background: Americans of African ancestry (AA) show disparities in ischemic stroke when compared to Euro-Americans: two-fold more stroke prevalence and mortality, greater stroke disability and post-stroke complications, and slower recovery. Thromboembolic ischemic stroke involves platelet hyperactivity, which stroke-related oxidative stress promotes. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-guanosine 3',5'-cyclic monophosphate (cGMP) pathway regulates vascular and platelet homeostasis. Oxidized (Fe 3+ ) sGC heme is NO-insensitive, lessening cGMP generation for vascular relaxation and platelet quiescence. Cytochrome b5 reductase 3 (CYB5R3) is a methemoglobin reductase with multiple functions, including NADH-supported redox cycling (Fe 3+ -> Fe 2+ ) of sGC heme. More than 40 variants of CYB5R3 exist. The T117S variant (threonine to serine substitution) has high frequency expression (23%) almost exclusively in AA persons (<1% other races) and has hypomorphic activity (~50%). Clinical data reveal more ischemic stroke (baseline NIHSS P =0.018) and delayed recovery (90 days MRS P =0.10) in carriers of T117S versus WT CYB5R3. We thus hypothesized that T117S increases risk for ischemic stroke and poor outcomes via hyperreactive platelets. Methods: Crispr-generated T117S mice (C57Bl6 background) and wild type (WT) mice (male, aged 3 mo) underwent transient MCAO (50 min occlusion, 24 hr reperfusion) +/- 3-week pretreatment with Bay54-6544, an NO-independent drug that activates sGC with oxidized (Fe 3+ ) or apo-heme. At sacrifice, blood was collected (5% EDTA) and brains stained with triphenyltetrazolium chloride to assess infarct volume. Platelets isolated by gentle serial centrifugation (110 g for plasma separation, 600 g for platelet separation) were probed by Western blot for platelet activation. Results: Untreated T117S mice displayed larger infarct volume ( P =0.0171) and platelet activation than WT mice (vWF P= 0.005, CD36 P= 0.051, EPO P= 0.055, STIM1 P =0.038). Bay54-6544 equalized T117S and WT infarct volumes ( P =0.684) and decreased post-stroke T117S platelet activation (vWF P= 0.0002, CD36 P= 0.038, EPO P= 0.001, STIM1 P <0.0001). We conclude that ischemic stroke activates platelets more in T117S mice versus WT, which Bay54-6544 pretreatment disrupts. For AA persons, T117S may predict increased risk for ischemic stroke. Prophylactic use of Bay54-6544 may lessen platelet contributions to ischemic stroke in T117S carriers and improve recovery times.

The glutathione S-transferase BnGSTU12 enhances the resistance of Brassica napus to Sclerotinia sclerotiorum through reactive oxygen species homeostasis and jasmonic acid signaling.

Sclerotinia sclerotiorum is a severe disease that affects rapeseed (Brassica napus), resulting in significant yield losses. In previous study, we identified the candidate GLUTATHIONE S-TRANSFERASE (GST) gene, BnGSTU12, associated with sclerotiorum stem resistance and the expression levels of BnGSTU12 in resistant lines were higher than that in susceptible lines. We analyzed the function of the BnGSTU12 during S. sclerotiorum infection in this study. BnGSTU12 expression was induced by S. sclerotiorum, with a strong increase 24h after onset of infection. Transgenic functional analysis indicated that overexpression of BnGSTU12 in Arabidopsis thaliana and B. napus enhanced resistance to S. sclerotiorum, whereas BnGSTU12 silencing decreased S. sclerotiorum resistance. The inoculated BnGSTU12-OE A. thaliana and B. napus plants showed higher antioxidant enzyme activity and lower H2O2 contents than the wild type. As BnGSTU12 was rapidly induced by the phytohormones salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), we investigated the involvement of the JA and SA pathways in GSTU12-mediated S. sclerotiorum resistance. JA content was higher in infected BnGSTU12-OE plants than in the wild type, whereas their SA contents were comparable. In addition, the expression levels of JASMONATE RESISTANT (JAR) involved in JA-Ile biosynthesis and those of JA-responsive genes were higher, the expression of JAZ gene repressing JA signaling was less in OE plants than WT after 12 and 24h inoculation with S. sclerotiorum. Our results show that BnGSTU12 enhances resistance to S. sclerotiorum through ROS homeostasis and JA signaling.

Differential structural characteristics, physicochemical properties, and calcium-binding capabilities of annexin A2 wild-type versus E53A, E96A, D162A, E247A and D322A mutants.

Annexin A2 (ANXA2) is a Ca2+-dependent multifunctional protein containing five Ca2+-binding domains, but their functional significance and difference remain unclear. Herein, glutamic acid (E) or aspartic acid (D) in five Ca2+-binding domains of canine ANXA2 (98.82% and 96.76-99.41% identical to ANXA2 from human and other mammals, respectively) was substituted by alanine (A) using site-directed mutagenesis. Recombinant ANXA2 wild-type (WT) and E53A, E96A, D162A, E247A and D322A mutants were constructed and expressed using a bacterial expression system followed by high-affinity purification using nickel-nitrilotriacetic acid (Ni-NTA) matrix. Efficacies of their expression and purification were confirmed by SDS-PAGE and Western blotting. Their amino acid sequences were verified by nanoLC-ESI-Qq-TOF tandem mass spectrometry. ATR-FTIR spectroscopy revealed that their secondary structure significantly differed (α-helix decreased but random coil increased in all mutants). Analyses of physicochemical properties revealed that molecular weight slightly decreased, whereas isoelectric point, aliphatic index, grand average of hydropathicity, electrostatic potential and molecular hydrophobicity potential slightly increased in all the mutants compared with WT. Interestingly, Ca2+-binding capability of these mutants (particularly E96A and D322A) significantly decreased from that of WT. In summary, secondary structure, physicochemical properties, and Ca2+-binding capability of E53A, E96A, D162A, E247A and D322A mutants of ANXA2 significantly differed from its WT, consistent with the loss of negatively charged E/D. In particular, E96A and D322A exhibited the lowest Ca2+-binding capability. These data and recombinant proteins would be useful for further investigations of the Ca2+-dependent functions of individual Ca2+-binding domains in ANXA2.

Abstract TMP113: Sex differences in the effects of social isolation on behavioral and metabolic parameters in a model of cerebral amyloid angiopathy

Introduction: The prevalence of social isolation (SI) increases with aging and contributes to poor health outcomes and increased risk of cognitive impairment, especially in Aβ-related diseases. Cerebral amyloid angiopathy (CAA) is characterized by amyloid β (Aβ) deposition in the cortical and leptomeningeal vessels, and is a leading cause of hemorrhagic stroke and age-related cognitive impairment. How social isolation affects vascular amyloid deposition, cognitive decline and social behavior in CAA is unknown. Methods: TgSwDI mice carrying Swedish, Dutch, and Iowa mutations of human amyloid precursor protein were used as a CAA model. Male and female C57BL/6 wildtype (WT) and CAA mice were randomly assigned to pair housing (PH) or SI at 3 months of age. Affective and social behaviors were assessed at baseline and every 3 months thereafter using the tail suspension test (TST) and three-chamber social interaction test. At 9 months post-SI, cognitive behaviors and general well-being were measured using fear conditioning (FC) and the nesting test, respectively. Body weights were recorded, and white adipocyte size was measured using immunostaining. Results: SI led to increased immobility in the TST at 3- and 6-months post-SI in both male and female WT and CAA mice, suggesting depressive-like behavior (n=5-8/grp, p<0.05). Isolated CAA mice displayed greater cognitive deficits in FC, while only male WT SI mice showed impaired cognition (n=5-12/grp, p<0.005). Sex differences in social behavior were observed in isolated CAA mice. Specifically, at 6 months post-SI, female CAA mice displayed deficits in sociability and social novelty (n=4-9/grp, p<0.05), which was not seen in male CAA mice. SI negatively impacts the general well-being in both male WT and CAA SI animals (n=5-12/grp, p<0.005). Preliminary findings showed increased white adipocyte size in male WT SI mice with a similar trend in male CAA SI animals, pointing to potential metabolic effects of isolation. Conclusion: SI induced cognitive decline in both male and female CAA mice. Behavioral differences between sexes were noted in CAA mice including nesting ability, sociability, and depressive-like behaviors. Further research is needed to determine the cellular and molecular mechanism underlying SI-induced metabolic changes in CAA.

Reactive carbonyl species function downstream of reactive oxygen species in chitosan-induced stomatal closure.

An elicitor, chitosan (CHT), induces stomatal closure in plants, which is accompanied by salicylhydroxamic acid (SHAM)-sensitive peroxidases-mediated reactive oxygen species (ROS) production in guard cells. Reactive carbonyl species (RCS) function downstream of ROS in abscisic acid (ABA) and methyl jasmonate (MeJA) signalling in guard cells. However, the involvement of RCS in CHT-induced stomatal closure is still unknown. In this study, we used transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis thaliana 2-alkenal reductase (AER-OE tobacco) and Arabidopsis wild-type (WT) plants to investigate whether RCS is involved in CHT-induced stomatal closure. Chitosan-induced stomatal closure was inhibited in the tobacco AER-OE plants. In the WT tobacco and Arabidopsis plants, CHT-induced stomatal closure was inhibited by RCS scavengers, carnosine and pyridoxamine. Chitosan significantly increased RCS production in the WT tobacco and Arabidopsis, but in the tobacco AER-OE plants, chitosan did not increase significantly RCS accumulation. Moreover, neither the application of RCS scavengers to both WT plants nor scavenging RCS by AER-OE affected the CHT-induced ROS accumulation. However, treatment with a peroxidase inhibitor, SHAM, significantly inhibited CHT-induced RCS accumulation in WT tobacco and Arabidopsis plants. Taken together, these results suggest that RCS acts downstream of ROS production in CHT signalling in guard cells of A. thaliana and N. tabacum.

FcγRI plays a pro-inflammatory role in the immune response to Chlamydia respiratory infection by upregulating dendritic cell-related genes.

FcγRI, a pivotal cell surface receptor, is implicated in diverse immune responses and is ubiquitously expressed on numerous immune cells. However, its role in intracellular bacterial infections remains understudied. Wild-type (WT) and FcγRI knockout (FcγRI-KO) mice were inoculated intranasally with a specific dose of C. muridarum. Lung tissues were harvested for transcriptome sequencing, and flow cytometry was employed to validate bioinformatics immune infiltration analysis. Differentially expressed DC-associated genes were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses to elucidate their functions during infection. A PPI network was constructed to pinpoint crucial genes, and qPCR was utilized to confirm their expression changes. Additionally, we compared body weight, lung Chlamydia load, and pathological alterations between WT and FcγRI-KO mice post-infection to assess the effect of FcγRI on inflammation via gene regulation. Lastly, an mRNA-miRNA-lncRNA network was formulated to further probe the molecular mechanisms of FcγRI in C. muridarum infection. Post-C. muridarum infection, FcγRI-KO mice exhibited a notable decrease in DC infiltration and maturation, along with downregulated co-stimulatory molecules (CD40, CD80, CD86) in lung tissues. Differential gene analysis identified 26 differentially expressed DC-related genes implicated in DC proliferation, migration, and inflammatory responses. KEGG analysis revealed their close association with key immune pathways. The PPI network delineated two modules, with the top six genes in the pivotal cluster 1 (Ccl4, Il6, Ccl3, Ptgs2, Il 1α, Il7) being significantly downregulated in FcγRI-KO mice. A ceRNA network encompassing 12 miRNAs and 37 lncRNAs regulating four key genes (Ptgs2, Il1α, Il6, Il7) was also constructed. In C. muridarum respiratory infections, FcγRI facilitates DC infiltration and maturation, upregulates six pro-inflammatory genes (Ccl4, Il6, Ccl3, Ptgs2, Il1α, Il7), and exhibits a pro-inflammatory role. A key ceRNA network was formulated to unravel the underlying molecular mechanisms.

Synergistic effects of temperature and light on photoprotection in the model diatom Phaeodactylum tricornutum.

Diatoms dominate phytoplankton communities in turbulent waters, where light fluctuations can be frequent and intense. Due to this complex environment, these heterokont microalgae display remarkable photoprotection strategies, including a fast Non-Photochemical Quenching (NPQ). However, in nature, several abiotic parameters (such as temperature) can influence the response of photosynthetic organisms to light stress in a synergistic or antagonistic manner. Yet, the combined effects of light and these other drivers on the photosynthetic and photoprotective capacity of diatoms are still poorly understood. In this work, we investigated the impact of short-term temperature and light stress on the model diatom Phaeodactylum tricornutum, combining NPQ induction-recovery assays or light curves with a broad gradient of superimposed temperature treatments (5 to 35°C). We employed mutant lines deficient in NPQ generation (vde KO) or recovery (zep3 KO) and wild type. We found that temperature and light have a synergistic effect: lower temperatures limited both the photosynthetic capacity and NPQ, while the general photophysiological performance was enhanced with warming, up to a heat-stress limit (above 30°C). We discuss the temperature effects on NPQ induction and recovery and propose that these are independent from the energy requirements of the cells and result from altered xanthophyll cycle dynamics. Namely, we found that de-epoxidation activity strongly increases with temperature, outweighing epoxidation and resulting in a positive increase of NPQ with temperature. Finally, we propose that in a short-term time frame, temperature and light have a synergistic and not antagonistic effect, with a positive relationship between increasing temperature and NPQ.

Overexpression of the Vitis amurensis Ca2+-binding protein gene VamCP1 in Arabidopsis thaliana and grapevine improves cold tolerance.

Calcium ions (Ca2+) are important second messengers and are known to participate in cold signal transduction. In the current study, we characterized a Ca2+-binding protein gene, VamCP1, from the extremely cold-tolerant grape species Vitis amurensis. VamCP1 expression varied among organs but was highest in leaves following cold treatment, peaking 24 h after treatment onset. VamCP1 was found to localize to the plasma membrane and nucleus and the gene showed transcriptional autoactivation activity. Overexpression of VamCP1 in Arabidopsis thaliana and grapevine (V. vinifera) resulted in transgenic plants that were more tolerant to cold stress than the wild type. This correlated with reduced accumulation of reactive oxygen species (ROS), elevated activity of antioxidant enzymes and proline content, as well as lower levels of malondialdehyde and electrolyte leakage. Additionally, the expression of genes related to cold tolerance, including C-repeat binding factors (CBF) and cold-regulated (COR) genes, was higher in the transgenic lines. Taken together, our results indicate that overexpression of VamCP1 enhanced cold tolerance in plants by promoting the upregulation of genes related to cold tolerance and scavenging of excessive ROS. These findings provide a foundation for the molecular breeding of cold-tolerant grapevine.

A functional helix shuffled variant of the B domain of Staphylococcus aureus.

The B domain of protein A is a biotechnologically important three-helix bundle protein. It binds the Fc fragment of antibodies with helix 1/2 and the Fab region with helix 2/3. Here we designed a helix shuffled variant by changing the connectivity of the helices, in order to redesign the helix bundle, yielding altered helix-loop-helix properties. The new loops that generate the new connectivity were created in several protein libraries, and Fc binding variants were selected for a detailed biochemical characterization. We were able to create variants with Fc binding affinity at the same level as the wild type B but with significantly reduced thermal stability. The NMR structure proved that the overall three-dimensional structure was maintained not only in the helix shuffled variant but also points to some potential local differences to wild-type B, which could be the reason for the reduced thermal stability. Therefore, protein A is an example of an optimized structure being more important for stability than for function. Using the helix shuffled variant as a ligand on an affinity column facilitates a robust and straightforward purification of antibodies, but allows for a milder elution at less extreme pH. Therefore, the helix shuffled variant is a suitable ligand to purify more pH-sensitive antibodies.

Maize ZmWRKY71 gene positively regulates drought tolerance through reactive oxygen species homeostasis.

Drought stress severely affects plant growth and yield. The plant-specific WRKY transcription factors play an important role in regulating the plant response to abiotic stresses. In this study, we identified a group I WRKY gene from maize, designated ZmWRKY71. Real-time quantitative reverse transcription-PCR analysis revealed that ZmWRKY71 was predominantly expressed in the roots and was induced by drought. ZmWRKY71 was localized in the nucleus and showed transcriptional activity in yeast. Heterologous overexpression of ZmWRKY71 improved drought tolerance in yeast and Arabidopsis. Compared with the wild type, the overexpression lines showed a higher survival rate under drought stress with reduced malondialdehyde content and elevated antioxidant enzyme activities. In contrast, mutation of ZmWRKY71 in maize leads to increased sensitivity to drought stress, reduced survival, elevated concentrations of reactive oxygen species, and increased malondialdehyde content. RNA-sequencing analysis revealed that the expression patterns of genes associated with translation, membrane, and oxidoreductase activity pathways were altered under drought stress. Yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays confirmed that ZmWRKY71 was capable of directly binding to the W-box element in the promoter region of ZmPOD42 (Zm00001eb330550). Taken together, the results show that ZmWRKY71 positively regulates maize drought tolerance. This research enriches the drought tolerance gene pool for maize and provides a theoretical basis for maize drought tolerance breeding.

MLH1 gene promoter methylation status partially overlaps with CpG methylator phenotype (CIMP) in colorectal adenocarcinoma.

RAS/BRAF mutations, mismatch DNA repair complex deficiency (MMRd)/microsatellite instability (MSI), and CpG methylator phenotype (CIMP) are key molecular actors in colorectal carcinogenesis. To date, conflicting evidence about the correlations between these molecular features has been reported. A retrospectively selected cohort of 123 CRCs was divided into 3 groups based on the molecular characteristics: MMR proficient (MMRp)/BRAF p.V600E mutated (BRAFmut), MMRd/BRAFmut, and MMRd/BRAF wild type (BRAFwt). MLH1 promoter (pMLH1) methylation status was assessed by pyrosequencing. For 82 samples the CIMP phenotype was evaluated using the EpiTect® MethyLight kit. The MMRd/BRAFmut group showed a higher pMLH1 methylation rate compared to both the MMRd/BRAFwt and the MMRp/BRAFmut groups. Overall, the two MMRd groups had a higher methylation rate compared to the MMRp cases independently from the mutational status of BRAF (p-value <0.0001). The MMRd/BRAFmut group was characterized by a 90.0 % of CIMP high (CIMP-H) tumors of which 97.2 % were pMLH1 methylated. Instead, the MMRd/BRAFwt group presented 50.0 % of CIMP-H adenocarcinomas. Our study demonstrates that pMLH1 hypermethylation, MMRd, BRAFmut and CIMP phenotype do not completely overlap in CRC. These findings further refine the knowledge on the molecular landscape of CRC and may have critical implications also for the clinical management of the disease.

The losses of Lem2 and Bqt4 exhibit similar impacts on intracellular movement dynamics in fission yeast.

Quantifying movement dynamics inside a cell provides a deeper understanding of cellular functions. The 3-dimensional live observation has been widely applied to movement dynamics research. Fission yeast is an ideal model organism for studying cellular movement dynamics. In this study, we developed a novel method to quantify intracellular movement in wild-type cells, using a vector originating from the cell center as the movement reference. This method obtained more movement information, including movement direction, compared to the previously reported method, which used a single point as the movement reference. Using this new method, we quantified intracellular movement in wild type, bqt4Δ, and lem2Δ cells across various parameters. We characterized the nature of intracellular movement in wild-type cells and revealed that the losses of Bqt4 and Lem2, two inner membrane proteins, impact intracellular movement in a similar pattern, although distinct differences were also observed. These findings offer new insights into the study of the overlapping and distinct functions of Bqt4 and Lem2. This study provides a novel method for evaluating the intracellular movement dynamics, contributing to the understanding of intracellular movement nature, biophysical properties, and the evaluation of genes or proteins from an intracellular movement perspective.

Semi-Rational Engineering of Glucosamine-6-Phosphate Deaminase for Catalytic Synthesis of Glucosamine from D-Fructose

Glucosamine (GlcN), as one of the important derivatives of D-glucose, is formed by the substitution of the hydroxyl group at position 2 of glucose with an amino group. As a bioactive amino monosaccharide, GlcN is known for its various biological effects, including immune enhancement, antioxidant, anti-inflammatory, hepatoprotective, joint pain relief, and alleviation of osteoporosis. These properties highlight the broad applications of GlcN and its derivatives in pharmaceuticals, cosmetics, food production, and other fields, underscoring their promising prospects. Thus, the efficient industrial production of GlcN is gaining increasing attention as well. Here, we report a novel biosynthetic method for GlcN, utilizing engineered Escherichia coli expressing glucosamine-6-phosphate deaminase (GlmD) to directly convert D-fructose into GlcN. The best mutant screened using the Morgan-Elson colorimetric method is the triple mutant G42S/G43C/G136T (designated as GlmD-ZH11), which exhibits approximately 21 times higher catalytic activity towards D-fructose compared to the wild type. Using the purified enzyme of GlmD-ZH11 in shaken flask fermentation for six hours, we achieved a conversion rate of 72.11% from D-fructose to GlcN. To further elucidate the mechanism behind the enhanced activity of the GlmD-ZH11 mutant, we conducted hydrogen bond network analysis to investigate the hydrogen bond interactions between the mutant and fructose. Additionally, we performed molecular dynamics simulations to study the RMSD and RMSF curves of the mutant. The results indicate that the protein structure of the mutant ZH11 is more stable and binds more tightly to the substrate. Calculations of the solvent-accessible surface area and binding free energy suggested that Thr41, Ser42, Asp72, Gly137, and Ala145 may be key amino acid residues in the catalytic process of ZH11. Finally, based on these findings and the catalytic mechanism of the wild type, we hypothesized a potential catalytic reaction mechanism for the ZH11 mutant.

Time of day dependent reduction in stroke infarct volume by the Reverb agonist SR9009 in mice

Ischemic stroke leads to disability and death worldwide and evidence suggests that stroke severity is affected by the time dimension of the stroke. Rev-Erbα regulates the core circadian clock through repression of the positive clock element Bmal1. However, it remains unclear if a Rev-Erbα agonist (SR9009) alleviates stroke pathology in mice. We found that stroke reduces the level of Rev-Erbα and elevates neuroinflammation and stroke severity at zeitgeber time (ZT) ZT06. Therefore, we hypothesized that SR9009 treatment may reduce neuroinflammation and stroke severity in a mouse suture occlusion model. At 12 to 14 weeks, C57BL/6 J (Wild Type, n = 5–10 mice/group) mice were randomly assigned to undergo MCAO stroke for 60 min at either zeitgeber time ZT06 (MCAO-ZT06-sleep phase) or ZT18 (MCAO-ZT18-awake phase). Stroked mice were treated with SR9009 (100 mg/kg) or vehicle at 1 h and 24 h after MCAO. After forty-eight hours of stroke, TTC staining, Western blot, and qRT-PCR were performed. We found that SR9009 treatment alleviates neuroinflammation and infarct volume by Rev-Erb remodeling in ZT06 stroke mice but not in ZT18 stroke mice. Additionally, monocytic and neutrophilic NLRP3 as well as brain NLRP3 levels were reduced by SR9009 treatment in ZT06 stroke though no effects were observed at ZT18 stroke. SR9009 also reduced TNFα expression and increased IL-10 expression in blood and brain in ZT06 stroke mice and no differences were observed at ZT18. There were no significant effects of SR9009 on neurological deficit score and sensorimotor function at ZT06 or ZT18 at 48 h. Our study demonstrates that SR9009 treatment reduces stroke volume, circulating immune response, circadian expression, and that the protection was circadian- and treatment time-dependent.

Up-regulation of Melanophilin (MLPH) gene during avian adipogenesis and decreased fat pad weights with adipocyte hypotrophy in MLPH knockout quail.

Advanced genetic and nutritional strategies aimed at modulating fat deposition can significantly reduce production costs and enhance profitability in the poultry industry. Melanophilin (MLPH) is recognized as a key gene regulating pigmentation as shown by diluted hair and feather coloration in MLPH mutant animals, including avian models. However, the effects of MLPH during fat accretion have not been studied yet. Therefore, the objectives of the current study are to measure the temporal expression of the MLPH gene during the adipocyte differentiation in vitro and in vivo and to investigate the effect of MLPH loss on fat accretion and adipocyte sizes in vivo using MLPH knockout quail model. The current in vitro studies reveal that MLPH gene expression levels were considerably elevated during adipogenesis in avian cells [101-fold in DF-1, 28.5-fold in chicken embryonic fibroblasts (CEF) and 4-fold in quail embryonic fibroblasts (QEF), compared to the undifferentiated cells of each cell type, p < 0.05]. In addition, fractionated fat cells (FC) showed increased expression levels of MLPH (5.7-fold, p < 0.05) compared to stromal-vascular cells (SVC). Using the MLPH knockout quail, disruption of the MLPH gene resulted in significantly reduced body weight (BW) and subcutaneous fat (S. Fat) pad weights compared to the wild type (WT) (p < 0.05). Further analysis through sectioning and staining of the fat tissues revealed that the mutation in Rab binding domain (RBD) of quail MLPH resulted in decreased fat cell sizes (p < 0.01). Overall, our data clearly demonstrated that MLPH can be a potential adipogenic marker gene, and MLPH may be associated with fat accretion in the gene edited quail model, highlighting the important role of MLPH in adipogenesis.

Thymidine phosphorylase participates in platelet activation and promotes inflammation in rheumatoid arthritis.

The elevated risk of cardiovascular disease (CVD) associated with inflammatory rheumatic diseases has long been recognized. Patients with established rheumatoid arthritis (RA) have a higher mortality rate compared to the general population due to abnormal platelet activation. Thymidine phosphorylase (TYMP) plays a crucial role in platelet activation and thrombosis, following bridging the link between RA and CVD. Data from Gene Expression Omnibus (GEO) database exhibited that TYMP levels were highly expressed in synovial tissues, immune cells, and whole blood of RA patients especially those with high levels of inflammation. Platelet count (PLT) and plateletcrit (PCT) were positively correlated with the severity of inflammation in rheumatoid arthritis while platelet distribution width (PDW) and mean platelet volume (MPV) were adverse. Levels of CD62P and TYMP in platelets of patients with active RA were significantly elevated compared to patients in the inactive phase. In vivo exepriments showed that reducing TYMP expression levels of platelets could relieve inflammation in Adjuvant-Induced Arthritis (AIA) mice. Platelet activation was significantly elevated in AIA model mice, along with increased levels of intracellular calcium (Ca2+), reactive oxygen species (ROS), and decreased Mitochondrial Membrane Potential (ΔΨm). However, treatment with Tipiracil hydrochloride (TPI) or the utilization of Tymp-/- mice reversed these effects. In vitro stimulation of wild type (WT) mouse platelets with tumor necrosis factor-alpha (TNF-α) promoted platelet activation, elevated levels of intracellular Ca2+as well as ROS while decreased ΔΨm. Platelets of WT mice treated with TPI or platelets of Tymp-/- mice exhibited the adverse results. Our study illustrates the vital role of TYMP in promoting RA inflammation and platelet activation, suggesting that TYMP may be a potential therapeutic target for RA.

Correlation Between PIK3R1 Expression and Cell Growth in Human Breast Cancer Cell Line BT-474 and Clinical Outcomes.

While mutations in the PIK3CA gene play important roles in human breast carcinogenesis, PIK3R1 gene alterations are recognized as actionable mutations for clinical cancer treatment. We aimed to elucidate the role of PIK3R1 in cell proliferation on breast carcinoma and to correlate the PIK3R1 expression with patients' outcome using human tumor tissue arrays. Using human BT-474 (estrogen receptor (ER)+/human epidermal growth factor receptor 2 (HER2)-high) breast carcinoma cell line as in vitro model, the role of PIK3R1 in cell proliferation was elucidated by knock-down of the PIK3R1 gene (ΔPIK3R1) in this cell line. Between January 2000 to December 2015, the records of a cohort of 440 patients in our hospital were retrospectively reviewed, including patients' survival. The correlations between PIK3R1 expression and patient prognosis, such as overall survival (OS) and disease-free survival (DFS), were elucidated by human breast cancer tumor tissue array immunostaining. After the PIK3R1 gene was silenced in the BT-474 line, there was an increased cell number and a decrease in the G0G1-fraction, and increased S-fraction and the S+G2M-fraction for the ΔPIK3R1-BT-474 cell line, as compared to their cell wild type (WT) line. Western blot analysis showed that decreased PIK3R1 protein levels were accompanied by an increase of the p-AKT and p-mTOR proteins in the ΔPIK3R1-BT-474 cell line, compared to the equivalent WT line. Using a human tumor tissue array, patients with high-expressed PIK3R1 protein had better outcomes in terms of DFS and OS, compared to those with low-expressed PIK3R1 protein, when breast cancer was at an early stage (stage I/II), but not across all stages of breast cancer in human patients. We concluded that downregulated PIK3R1 in BT-474 cells resulted in an increased cell growth and upregulated AKT-mTOR signaling. Clinically, the high-expressed PIK3R1 protein in tumors correlates positively with patients' outcome in stage I and II breast cancer.