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Resistance to KRAS therapy in pancreatic ductal adenocarcinoma (PDAC) involves cellular plasticity, particularly the epithelial-to-mesenchymal transition (EMT), which poses challenges for effective targeting. Chronic pancreatitis, a known risk factor for PDAC, elevates TGFβ levels in the tumor microenvironment (TME), promoting resistance to KRAS therapy. Mechanistically, TGFβ induces the formation of a novel protein complex composed of SMAD3, SMAD4, and the nuclear factor NFAT5, triggering EMT and resistance by activating key mediators such as S100A4. Inhibiting NFAT5 attenuates pancreatitis-induced resistance to KRAS inhibition and extends mouse survival. Additionally, TGFβ stimulates PDAC cells to secrete CCL2, recruiting macrophages that contribute to KRAS bypass through paracrine S100A4. Our findings elucidate the role of TGFβ signaling in EMT-associated KRAS therapy resistance and identify NFAT5 as a druggable target. Targeting NFAT5 could disrupt this regulatory network, offering a potential avenue for preventing resistance in PDAC.

ABSTRACTSETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETXresult in either of two distinct neurodegenerative disorders. SETXdominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.Abbreviations:3ʹREADS: 3ʹ region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A1; BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5ʹ-3ʹ exoribonuclease 2.

The article presents application of homo polymer of dodecyl methacrylate (DDMA) and its copolymers with vinyl acetate (VA) as multifunctional additives for lubricant formulation. Homo polymer of DDMA and five copolymers of DDMA with VA at different molar ratios were synthesized by free radical polymerization method using azobisisobutyronitrile (AIBN) as initiator. The characterization of the polymers was carried out through FTIR, NMR and GPC (gel permeable chromatography) analysis. The performance of all the polymers as viscosity index improver (VII) or viscosity modifier and pour point depressant (PPD) additive in two different base oils (mineral) were evaluated. The mechanism of action of the polymers as pour point depressant was studied by photo micrographic analysis. Rheological study of the formulated lubricant was also carried out and reported. The thermal stability of the polymers was determined by thermogravimetric analysis (TGA). It was found that thermal stability, VI and molecular weights of copolymers are higher than the homopolymer which showed better PPD property.

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3′UTRs and/or coding sequences from a single gene. Here, using 3′ region extraction and deep sequencing (3′READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogasterundergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3′UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3′UTRs and in introns, consistent with the “first come, first served” model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogasterand reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Alternative polyadenylation (APA) is a widespread mechanism that generates mRNA isoforms with distinct properties. Here we have systematically mapped and compared cleavage and polyadenylation sites (PASs) in two yeast species, S. cerevisiaeand S. pombe. Although >80% of the mRNA genes in each species were found to display APA, S. pombeshowed greater 3′ UTR size differences among APA isoforms than did S. cerevisiae. PASs in different locations of gene are surrounded with distinct sequences in both species and are often associated with motifs involved in the Nrd1-Nab3-Sen1 termination pathway. In S. pombe,strong motifs surrounding distal PASs lead to higher abundances of long 3′ UTR isoforms than short ones, a feature that is opposite in S. cerevisiae. Differences in PAS placement between convergent genes lead to starkly different antisense transcript landscapes between budding and fission yeasts. In both species, short 3′ UTR isoforms are more likely to be expressed when cells are growing in nutrient-rich media, although different gene groups are affected in each species. Significantly, 3′ UTR shortening in S. pombecoordinates with up-regulation of expression for genes involved in translation during cell proliferation. Using S. pombestrains deficient for Pcf11 or Pab2, we show that reduced expression of 3′-end processing factors lengthens 3′ UTR, with Pcf11 having a more potent effect than Pab2. Taken together, our data indicate that APA mechanisms in S. pombeand S. cerevisiaeare largely different: S. pombehas many of the APA features of higher species, and Pab2 in S. pombehas a different role in APA regulation than its mammalian homolog, PABPN1.

Mechanisms underlying neuropathic pain (NP) are complex with multiple genes, their interactions, environmental and epigenetic factors being implicated. Transcriptional changes in the trigeminal (TG) and dorsal root (DRG) ganglia have been implicated in the development and maintenance of NP. Despite efforts to unravel molecular mechanisms of NP, many remain unknown. Also, most of the studies focused on the spinal system. Although the spinal and trigeminal systems share some of the molecular mechanisms, differences exist. We used RNA-sequencing technology to identify differentially expressed genes (DEGs) in the TG and DRG at baseline and 3 time points following the infraorbital or sciatic nerve injuries, respectively. Pathway analysis and comparison analysis were performed to identify differentially expressed pathways. Additionally, upstream regulator effects were investigated in the two systems. DEG (differentially expressed genes) analyses identified 3,225 genes to be differentially expressed between TG and DRG in naïve animals, 1,828 genes 4 days post injury, 5,644 at day 8 and 9,777 DEGs at 21 days postinjury. A comparison of top enriched canonical pathways revealed that a number of signaling pathway was significantly inhibited in the TG and activated in the DRG at 21 days postinjury. Finally, CORT upstream regulator was predicted to be inhibited in the TG while expression levels of the CSF1 upstream regulator were significantly elevated in the DRG at 21 days postinjury. This study provides a basis for further in-depth studies investigating transcriptional changes, pathways, and upstream regulation in TG and DRG in rats exposed to peripheral nerve injuries.

The Equatorial Ionization Anomaly (EIA) is characterized by strong ionospheric gradients that complicate ionospheric modeling and mitigation of ionospheric impact on Global Navigation Satellite System applications. In this study, the variation of EIA crest locations as a function of longitude and solar radio flux index F10.7 has been derived and modeled. Seven years (2013–2019) of ionospheric NmF2 and in‐situ ion density data from Constellation Observing System for Meteorology Ionosphere and Climate and Swarm‐A satellites have been used. We find that the distance between the northern and southern crest of EIA grows slightly with increase of solar flux. The position of the crests of EIA follows a wave number 4 structure with peak values at around −130°, 0°, and 100°E. At the altitude of Swarm‐A (∼460 km), the equatorial crests are found closer to the geomagnetic dip equator than they are at the peak density height hmF2 (∼310 km) which may be due to the spatial variations of geomagnetic field strength. Accordingly, two Crest Position climatological Models have been developed, one for the F2 Peak height (CPM_F2P) and the other one for Swarm‐A height (CPM_SAH). At higher altitudes the crests of Equatorial Ionization Anomaly (EIA) are found closer to the dip equator than the crests at lower altitudeThe positions of the EIA crests show a weak linear correlation with solar radio flux (F10.7)Empirical models for the position of EIA crests are developed At higher altitudes the crests of Equatorial Ionization Anomaly (EIA) are found closer to the dip equator than the crests at lower altitude The positions of the EIA crests show a weak linear correlation with solar radio flux (F10.7) Empirical models for the position of EIA crests are developed

Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type–specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice.

ABSTRACTThe simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

Over the last 20 years, a large number of instruments have provided plasma density measurements in Earth's topside ionosphere. To utilize all of the collected observations for empirical modeling, it is necessary to ensure that they do not exhibit systematic differences and are adjusted to the same reference frame. In this study, we compare satellite plasma density observations from Gravity Recovery and Climate Experiment (GRACE), Constellation Observing System for Meteorology, Ionosphere, and Climate (COSMIC), CHAllenging Minisatellite Payload (CHAMP), Swarm, and Communications/Navigation Outage Forecasting System (C/NOFS) missions. Electron densities retrieved from GRACE K‐Band Ranging (KBR) system, previously shown to be in excellent agreement with incoherent scatter radar (ISR) measurements, are used as a reference. We find that COSMIC radio occultation (RO) densities are highly consistent with GRACE‐KBR observations showing a mean relative difference of <2%, and therefore no calibration factors between them are necessary. We utilize the outstanding three‐dimensional coverage of the topside ionosphere by the COSMIC mission to perform conjunction analysis with in situ density observations from CHAMP, C/NOFS, and Swarm missions. CHAMP measurements are lower than COSMIC by ∼11%. Swarm densities are generally lower at daytime and higher at nighttime compared to COSMIC. C/NOFS ion densities agree well with COSMIC, with a relative bias of ∼7%. The resulting cross‐calibration factors, derived from the probability distribution functions, help to eliminate the systematic leveling differences between the data sets, and allow using these data jointly in a large number of ionospheric applications. A systematic comparison of the plasma density data from CHAMP, C/NOFS, GRACE, COSMIC, and Swarm missions is performedElectron densities retrieved from COSMIC‐RO agree well with GRACE‐KBR observations showing a relative difference of less than 2%Intercalibration factors, allowing to eliminate the systematic offsets between the considered data sets, are presented A systematic comparison of the plasma density data from CHAMP, C/NOFS, GRACE, COSMIC, and Swarm missions is performed Electron densities retrieved from COSMIC‐RO agree well with GRACE‐KBR observations showing a relative difference of less than 2% Intercalibration factors, allowing to eliminate the systematic offsets between the considered data sets, are presented

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2, and VP3. These capsid proteins have a common amino acid sequence, being expressed from different initiation codons on the same open reading frame. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but their functions are poorly understood. To investigate the role(s) of the capsid proteins in capsid formation, we used a baculovirus protein expression system to produce virus-like particles (VLPs). We found that varying the ratios of VP2 and VP3 did not affect VLP formation. Further, their physical properties were equivalent to those of empty wild-type particles. The function of VP3 was studied further by fusing a peptide tag, FLAG, to its N-terminus. This chimeric viral protein, in combination with VP2, could assemble into VLPs, indicating that the chimerism of VP3 did not affect VLP formation. Although the monomeric native form of the FLAG-VP3 chimera could react with anti-FLAG antibody, VLP containing the chimeric VP3 could not, suggesting that the N-terminal region of VP3 is located inside the VLP. These observations indicate that it may be possible to utilize AAV VLP as vectors of a broad range of drugs since fusion of the VP3 N-terminus with defined molecules could impose distinct physical properties onto the internal environment of the VLP.

ABSTRACTAdeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosisthat is caused by a reversible frameshift mutation in the M. tuberculosisorngene. A frameshift mutation in ornproduces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same ornmutation in a low-level EMB-resistant double embB-aftAmutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml.

Most eukaryotic genes are transcribed into mRNAs with alternative poly(A) sites. Emerging evidence suggests that mRNA isoforms with alternative poly(A) sites can perform critical regulatory functions in numerous biological processes. In recent years, a number of strategies utilizing high‐throughput sequencing technologies have been developed to aid in the identification of genome‐wide poly(A) sites. This unit describes a modified protocol for a recently published 3′READS (3′ region extraction and deep sequencing) method that accurately identifies genome‐wide poly(A) sites and that can be used to quantify the relative abundance of the resulting 3′ mRNA isoforms. This approach minimizes nonspecific sequence reads due to internal priming and typically yields a high percentage of sequence reads that are ideally suited for accurate poly(A) identification. © 2015 by John Wiley & Sons, Inc.