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The valorization of wood waste as a sustainable bacterial feedstock for the production of Polyhydroxybutyrate (PHB) is explored in this study, aiming to provide an environmentally friendly alternative to conventional plastics. Wood waste, treated with 4% sulfuric acid, served as the carbon source for isolating bacteria from Jalandhar waste streams, with the strain Klebsiella sp. MK3 identified as the most effective in PHB production after 16s rRNA sequencing. Analytical methods including the Molisch test, DNS, and sugar utilization tests confirmed sugar presence and consumption by the bacterial isolate. Media optimization using Design Expert 12.0 utilized a quadratic model, achieving a robust fit with an R² value of 98.6%. Optimization via Plackett-Burman design and response surface methodology enhanced PHB yield to 4.37 mg/mL, a significant increase over previous benchmarks. This yield was achieved under optimal conditions of 1.7% carbon concentration, 0.105% nitrogen concentration, and a constant temperature of 37 °C. Qualitative analysis of PHB by UV-Vis spectroscopy, FTIR, and NMR confirmed its purity and composition. The study highlights the potential of wood waste and wastewater as substrates for cost-effective PHB production, with significant applications in packaging, agriculture, medicine, and more, thus promoting reduced reliance on non-renewable resources and advancing sustainability goals.

Multiple p38 MAP kinase inhibitors have been developed for the treatment of inflammatory diseases such as rheumatoid arthritis, but their effectiveness has been limited due to toxicity and tachyphylaxis, leading to a lack of clinical benefit. Efforts have been made to circumvent this limitation by targeting individual substrates downstream of p38, including MK2 and MK5. This approach has failed to yield clinical benefit despite preclinical evidence of a therapeutic effect. We hypothesized that there is redundancy in the MAPK activating kinase family that would necessitate blocking multiple kinases to sufficiently impact inflammatory processes. We used heterobifunctional protein degraders that either specifically degraded MK2 selectively or degraded MK2/3/5 simultaneously to test the hypothesis, in addition to genetic approaches to enable knockdown. In human PBMCs, elimination of MK2/3/5 with heterobifunctional degraders resulted in full reduction of TLR4 or TLR7/8 induced TNFα, whereas MK2-specific degradation only attenuated TNFα biosynthesis. In contrast, both specific MK2 degradation and broad MK2/3/5 degradation inhibited TGF-β-induced collagen production in human fibroblasts. This observation was consistent with genetic deletions of MK2, MK3 and MK5 (singly and in combination) whereby single deletion of MK2, MK3 or MK5 attenuated lipopolysaccharide (LPS) induced TNFα production and had no effect on R848-induced TNFα production. Double deletion of MK2 and MK3 or MK2 and MK5 or MK2/3/5 triple deletion had a significantly greater effect on TNFα production regardless of stimulus. The combined data suggest cooperativity between MK2 and either MK3 or MK5 for efficient, cell context-dependent modulation of inflammatory responses.

In poultry production, the laying rate is a critical economic trait, as high egg production significantly enhances profitability. Mitogen-activated protein kinase-activated protein kinase 3 (MK3) is a member of the mitogen-activated protein kinase (MAPK) family, which plays an important role in follicular development. Our previous RNA-seq analysis revealed that MK3 expression was significantly altered in the ovaries of laying hens exposed to normal versus light-deprivation conditions. Based on previous RNA-seq analysis of chicken ovaries, this study focused on the MK3 gene to explore its role in regulating apoptosis of follicular granulosa cells in laying hens. The results demonstrated that MK3 overexpression induced granulosa cell apoptosis by modulating the expression of key proliferation- and apoptosis-related genes, including FAS, Caspase3, BCL2, and C-myc. These findings were further validated using specific siRNA-mediated knockdown of MK3. Flow cytometry, CCK-8, and EdU assays consistently showed that MK3 facilitated apoptosis and inhibited granulosa cell proliferation. Additionally, dual-luciferase reporter assays revealed that the transcription factor WT1 bound to the MK3 promoter and enhanced its transcriptional activity. Mechanistically, MK3 regulated granulosa cell apoptosis through the TNF/P38 MAPK pathway. This conclusion was corroborated by treatment with the P38 inhibitor GS-444217 and specific siRNA targeting components of the pathway. In summary, MK3 promotes granulosa cell apoptosis in the follicles of laying hens, is transcriptionally regulated by WT1, and exerts its pro-apoptotic effects via the TNF/P38 MAPK pathway.

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

Six2 (Sine oculis homeobox 2), a homeodomain transcription factor, plays a crucial role in the regulation of mammalian nephrogenesis. It is also implicated in numerous biological functions, such as cell proliferation, apoptosis, and migration. However, the underlying regulatory mechanisms of Six2 remain largely unknown. In this study, we predicted that CRX, GATA1, HOXD8, and POU2F2 might target, binding to the promoter region of Six2 (∼2000 bp) by bioinformatics analysis. Among the four genes, the predicted binding sequence of GATA1 is most highly conserved across species. Luciferase assays demonstrated that knockdown of GATA1 decreased the activity of Six2 promoter and qPCR result of Six2 expression was in consistent with this in 293T cells. Mutation of GATA1 binding sites of mSix2 promoter led to obvious decrease of the mSix2 promoter activity. Furthermore, knockdown of GATA1 decreased Six2 expression in mk3 cells and increased cell apoptosis of mk3 and mk4 compared with corresponding control cells, but this up-regulation can be rescued by Six2 overexpression. Our findings indicated that GATA1 may be a potential regulator of Six2-maintained population of nephron progenitor cells.

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC 50  = 37.5 μM; K i  = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.

Background Gene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1). The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs), all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3 , a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription. Results Our current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the existence of MK-mediated negative feedback regulation on pERK. Conclusion We here identify and characterize important actors in a PRC1-dependent epigenetic signal/response mechanism, some of which appear to be nonspecific global responses, whereas others provide modular specificity. Our findings provide novel insight into a Polycomb-mediated epigenetic mechanism that dynamically controls gene transcription and support a direct link between PRC1 and cellular responses to changes in the microenvironment.

Murid herpesvirus 4 (MuHV-4) currently serves as a model for study of human gamma-herpesvirus pathogenesis. It codes for MK3 protein that similarly as K5 protein of Kaposi's sarcoma-associated herpesvirus are members of a family of structurally related viral immune evasion molecules possessing RING-CH finger domain with ubiquitin ligase activity. Murine herpesvirus 72 (MHV-72) isolated from the same species of free-living small rodent is considered as closely related to Murine herpesvirus 68 (MHV-68). Studies on MHV-72, identified dissimilarity from MHV-68 in the sequence of glycoprotein 150 [K. Macáková, J. Matis, I. Rezuchová, O. Kúdela, H. Raslová, M. Kúdelová, Virus Genes 26, 89-95 (2003)]. Murine herpesvirus 4556 (MHV-4556) is relatively new, till now, uncharacterised strain isolated from different murid species Apodemus flavicollis. We have therefore sequenced the MK3 gene of MHW-72 as well as of MHV-4556 to find out the evidence of their difference from that of MHV-68. We show here the unique nucleotide mutation in MHV-72 MK3 gene changing the codon at C-end of MK3 protein that was earlier predicted to function in interaction with TAP1/2. Furthermore, one from two nucleotide mutations found for MHV-4556 MK3 gene changed the codon that is localized at N-terminus of MK3 protein. MHV-4556-specific mutation was found within MK3 RING-CH finger domain known to be necessary for the ubiquitination of MHC class I proteins. Moreover, the latter established the new restriction site specific for MHV-4556.

Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed.