PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.

Programmed death ligand 1 (PD-L1, also called B7-H1) is an immune checkpoint protein that inhibits immune function through its binding of the programmed cell death protein 1 (PD-1) receptor. Clinically approved antibodies block extracellular PD-1 and PD-L1 binding, yet the role of intracellular PD-L1 in cancer remains poorly understood. Here, we discovered that intracellular PD-L1 acts as an RNA binding protein that regulates the mRNA stability of NBS1, BRCA1, and other DNA damage-related genes. Through competition with the RNA exosome, intracellular PD-L1 protects targeted RNAs from degradation, thereby increasing cellular resistance to DNA damage. RNA immunoprecipitation and RNA-seq experiments demonstrated that PD-L1 regulates RNA stability genome-wide. Furthermore, we developed a PD-L1 antibody, H1A, which abrogates the interaction of PD-L1 with CMTM6, thereby promoting PD-L1 degradation. Intracellular PD-L1 may be a potential therapeutic target to enhance the efficacy of radiotherapy and chemotherapy in cancer through the inhibition of DNA damage response and repair.

Evidence for massive methane hydrate destabilization during the penultimate interglacial warming.

The stability of widespread methane hydrates in shallow subsurface sediments of the marine continental margins is sensitive to temperature increases experienced by upper intermediate waters. Destabilization of methane hydrates and ensuing release of methane would produce climatic feedbacks amplifying and accelerating global warming. Hence, improved assessment of ongoing intermediate water warming is crucially important, especially that resulting from a weakening of Atlantic meridional overturning circulation (AMOC). Our study provides an independent paleoclimatic perspective by reconstructing the thermal structure and imprint of methane oxidation throughout a water column of 1,300 m. We studied a sediment sequence from the eastern equatorial Atlantic (Gulf of Guinea), a region containing abundant shallow subsurface methane hydrates. We focused on the early part of the penultimate interglacial and present a hitherto undocumented and remarkably large intermediate water warming of 6.8 °C in response to a brief episode of meltwater-induced, modest AMOC weakening centered at 126,000 to 125,000 y ago. The warming of intermediate waters to 14 °C significantly exceeds the stability field of methane hydrates. In conjunction with this warming, our study reveals an anomalously low δ13C spike throughout the entire water column, recorded as primary signatures in single and pooled shells of multitaxa foraminifers. This extremely negative δ13C excursion was almost certainly the result of massive destabilization of methane hydrates. This study documents and connects a sequence of climatic events and climatic feedback processes associated with and triggered by the penultimate climate warming that can serve as a paleoanalog for modern ongoing warming.

Krüppel-like factor 15 deficiency exacerbates osteoarthritis through reduced expression of peroxisome proliferator-activated receptor gamma signaling in mice.

OBJECTIVE: Krüppel-like zinc finger transcription factors (KLFs) play diverse roles in mammalian cell differentiation and development. In this study, we investigated the function of KLF15 in the progression of osteoarthritis (OA). METHODS: 0Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male wild-type control (WT) mice and cartilage-specific KLF15 knockout (KO) mice. Histological analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining were performed. Morphological changes were measured using microcomputed tomography. Six mice from each group were analyzed (total number of mice analyzed: 60). In vitro, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western blot analyses were performed. RESULTS: KLF15 KO DMM mice exhibited significant cartilage degradation compared to WT mice. According to the Osteoarthritis Research Society International cartilage OA-histopathology scoring system, the mean sum score in KLF15 KO mice was significantly higher than that in WT mice at 8 weeks after surgery. Immunohistochemistry results revealed KLF15 KO mice exhibited reduced peroxisome proliferator-activated receptor gamma (PPARγ) expression, increased pIKKα/ß, a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) 5, and Matrix metalloproteinases (MMP13) expression, and reduced Forkhead box O (FOXO1) and Light chain 3B (LC3B) expression. Inhibition of PPARγ phosphorylation accelerated the effects of interleukin (IL) 1ß-treatment in both KLF15 KO and WT chondrocytes, and activation of PPARγ expression canceled the IL1ß-induced catabolic effects. CONCLUSION: Our results indicated that the OA phenotype of KLF15 KO DMM mice was influenced by reduced PPARγ expression, including enhanced pIKKα/ß, ADAMTS5, and MMP13 expression, reduced autophagy, and increased apoptosis. KLF15 regulation may constitute a possible therapeutic strategy for the treating OA.

5H-benzo[c]fluorene derivative exhibits antiproliferative activity via microtubule destabilization.

Present study aimed at a single component cyclization of 2-benzylidene-1-tetralones for the preparation of 5H-benzo[c]fluorenes and their antiproliferative activity. This ring closure reaction underwent via reductive cyclization in the presence of a sodium borohydride-aluminium chloride system. Ten diverse 5H-benzo[c]fluorene derivatives were prepared and evaluated for antiproliferative activity against three human cancer cell lines by SRB assay. Four of these benzofluorenes exhibited significant antiproliferative effect with an IC50 < 10.75 µM. The best representative compound 21, exhibited IC50 against K562 leukemic cells at 3.27 µM in SRB assay and 7.68 µM in Soft agar colony assay. It exhibited a microtubule destabilization effect in tubulin kinetics and inhibited 82.9 % microtubule polymer mass at 10 µM concentration in Protein Sedimentation assay (Microtubule). Compound 21 exerted G0/G1 phase arrest in cell division cycle analysis in K562 cells. It also induced apoptosis in K562 cells via activation of Caspase cascade pathway. Furthermore, compound 21 also possessed anti-inflammatory activity by inhibiting TNF-α and IL-6 moderately. It exhibited significant in vivo efficacy and reduced K562 tumour in xenograft mice by 47 % at an 80 mg/kg oral dose. Further, it was found to be safe and well tolerable up to 1000 mg/kg in Swiss albino mice. Compound 21 needs to be optimized for better in vivo efficacy in rodent models for further development.

Microplastics destabilize lipid membranes by mechanical stretching.

Estimated millions of tons of plastic are dumped annually into oceans. Plastic has been produced only for 70 y, but the exponential rise of mass production leads to its widespread proliferation in all environments. As a consequence of their large abundance globally, microplastics are also found in many living organisms including humans. While the health impact of digested microplastics on living organisms is debatable, we reveal a physical mechanism of mechanical stretching of model cell lipid membranes induced by adsorbed micrometer-sized microplastic particles most commonly found in oceans. Combining experimental and theoretical approaches, we demonstrate that microplastic particles adsorbed on lipid membranes considerably increase membrane tension even at low particle concentrations. Each particle adsorbed at the membrane consumes surface area that is proportional to the contact area between particle and the membrane. Although lipid membranes are liquid and able to accommodate mechanical stress, the relaxation time is much slower than the rate of adsorption; thus, the cumulative effect from arriving microplastic particles to the membrane leads to the global reduction of the membrane area and increase of membrane tension. This, in turn, leads to a strong reduction of membrane lifetime. The effect of mechanical stretching of microplastics on living cells membranes was demonstrated by using the aspiration micropipette technique on red blood cells. The described mechanical stretching mechanism on lipid bilayers may provide better understanding of the impact of microplastic particles in living systems.

Osteoarthritis as a Systemic Disease Promoted Prostate Cancer In Vivo and In Vitro.

Osteoarthritis (OA) is increasing worldwide, and previous work found that OA increases systemic cartilage oligomeric matrix protein (COMP), which has also been implicated in prostate cancer (PCa). As such, we sought to investigate whether OA augments PCa progression. Cellular proliferation and migration of RM1 murine PCa cells treated with interleukin (IL)-1α, COMP, IL-1α + COMP, or conditioned media from cartilage explants treated with IL-1α (representing OA media) and with inhibitors of COMP were assessed. A validated murine model was used for tumor growth and marker expression analysis. Both proliferation and migration were greater in PCa cells treated with OA media compared to controls (p < 0.001), which was not seen with direct application of the stimulants. Migration and proliferation were not negatively affected when OA media was mixed with downstream and COMP inhibitors compared to controls (p > 0.05 for all). Mice with OA developed tumors 100% of the time, whereas mice without OA only 83.4% (p = 0.478). Tumor weight correlated with OA severity (Pearson correlation = 0.813, p = 0.002). Moreover, tumors from mice with OA demonstrated increased Ki-67 expression compared to controls (mean 24.56% vs. 6.91%, p = 0.004) but no difference in CD31, PSMA, or COMP expression (p > 0.05). OA appears to promote prostate cancer in vitro and in vivo.

ßTrCP1 promotes SLC35F2 protein ubiquitination and inhibits cancer progression in HeLa cells.

The solute carrier family 35 F2 (SLC35F2) belongs to membrane-bound carrier proteins that are associated with multiple cancers. The main factor that determines cancer progression is the expression level of SLC35F2. Thus, identifying the E3 ligase that controls SLC35F2 protein abundance in cancer cells is critical. Here, we identified ßTrCP1 interacting with and reducing the SLC35F2 protein level. ßTrCP1 signals SLC35F2 protein ubiquitination and reduces SLC35F2 protein half-life. The mRNA expression pattern between ßTrCP1 and SLC35F2 across a panel of cancer cell lines showed a negative correlation. Additionally, the depletion of ßTrCP1 accumulated SLC35F2 protein and promoted SLC35F2-mediated cell growth, migration, invasion, and colony formation ability in HeLa cells. Overall, we demonstrate that ßTrCP1 acts as a tumor suppressor by controlling SLC35F2 protein abundance in cancer cells. The depletion of ßTrCP1 promotes SLC35F2-mediated carcinogenesis. Thus, we envision that ßTrCP1 may be a potential target for cancer therapeutics.

Increasing antimicrobial susceptibility of MDR Salmonella with the efflux pump inhibitor 1-(1-Naphthylmethyl)-piperazine.

Salmonella is a widespread foodborne pathogen that can exhibit multidrug resistance (MDR; resistance to ≥3 antimicrobial classes). Therefore, the development of new preventative measures against MDR Salmonella is highly important. Bacterial antibiotic resistance is commonly mediated by efflux pumps. In this study, two compounds that block efflux pump activity, 1-(1-Naphthylmethyl)-Piperazine (NMP) and Phenylalanine-arginine ß-naphthylamide (PaßN), were tested with the antibiotic tetracycline to determine if a synergistic reduction in resistance could be achieved in tetracycline-resistant Salmonella. The efflux pump inhibitors (EPIs) reduced Salmonella resistance to tetracycline by 16 to 32-fold in several tetracycline resistant isolates. For example, the tetracycline minimum inhibitory concentration (MIC) for MDR Salmonella enterica serovar I 4,[5],12:i:- USDA15WA-1 (SX 238) was 256 µg/mL. However, in the presence of NMP (250 µg/mL), the MIC dropped to 8 µg/mL which is below the Clinical Laboratory Standards Institute (CLSI) breakpoint for tetracycline resistance in Salmonella (≥16 µg/mL). Confocal and transmission electron microscopy revealed NMP-mediated damage to Salmonella membranes at a higher concentration (1000 µg/mL), implying that the EPI disrupts membrane morphology which can lead to cell death; however, this effect was dependent on NMP concentration, as NMP blocked efflux activity with less of a membrane-disrupting effect at a lower concentration (250 µg/mL). These findings suggest that the use of EPIs can reduce the MIC of tetracycline and restore the effectiveness of the antibiotic against tetracycline-resistant Salmonella.

On the Role of Temperature in the Depolymerization of PET by FAST-PETase: An Atomistic Point of View on Possible Active Site Pre-Organization and Substrate-Destabilization Effects.

Enzyme FAST-PETase, recently obtained by a machine learning approach, can depolymerize poly(ethylene terephthalate) (PET), a synthetic resin employed in plastics and in clothing fibers. Therefore it represents a promising solution for the recycling of PET-based materials. In this study, a model of PET was adopted to describe the substrate, and all-atoms classical molecular dynamics (MD) simulations on apo- and substrate-bound FAST-PETase were carried out at 30 and 50 °C to provide atomistic details on the binding step of the catalytic cycle. Comparative analysis shed light on the interactions occurring between the FAST-PETase and 4PET at 50 °C, the optimal working conditions of the enzyme. Pre-organization of the enzyme active and binding sites has been highlighted, while MD simulations of FAST-PETase:4PET pointed out the occurrence of solvent-inaccessible conformations of the substrate promoted by the enzyme. Indeed, neither of these conformations was observed during MD simulations of the substrate alone in solution performed at 30, 50 and 150 °C. The analysis led us to propose that, at 50 °C, the FAST-PETase is pre-organized to bind the PET and that the interactions occurring in the binding site can promote a more reactive conformation of PET substrate, thus enhancing the catalytic activity of the enzyme.

Limited roles of Piezo mechanosensing channels in articular cartilage development and osteoarthritis progression.

OBJECTIVE: To investigate the role of Piezo1 and Piezo2 in surgically induced osteoarthritis (OA) in mice. DESIGN: Male conditional knockout (cKO) mice missing Piezo1 and Piezo2 in the joint using Gdf5-Cre transgenic mice were induced with post-traumatic OA by destabilization of the medial meniscus (DMM) of the right knee joint at 12 weeks of age. The severity of OA was histologically assessed at 24 weeks of age. OA-associated pain was evaluated by static weight bearing analysis. Additionally, articular chondrocytes isolated from cKO mice were exposed to fluid flow shear stress (FFSS) to evaluate the expression of OA-associated genes. RESULTS: Mice with conditional deletion of Piezo1 and Piezo2 showed normal joint development with no overt histological changes in the knee joint at 12 weeks and 24 weeks. DMM surgery induced moderate to severe OA in both control and cKO mice (median OARSI score: control, 4.67; cKO, 4.23, P = 0.3082), although a few cKO mice showed milder OA. Pain assessment by static weight-bearing analysis suggested Piezo ablation in the joint has no beneficial effects on pain. FFSS increased the expression of OA-related genes both in control and cKO mice to similar extents. CONCLUSION: Piezo1 and Piezo2 are not essential for normal joint development. Genetic ablation of Piezo channels did not confer evident protective effects on OA progression in mice. In vitro data suggests that different mechanotransducers other than Piezo channels mediate FFSS in mechanical stress-induced gene expression.

Activating M1 muscarinic cholinergic receptors induces destabilization of resistant contextual fear memories in rats.

Destabilization of previously consolidated memories places them in a labile state in which they are open to modification. However, strongly encoded fear memories tend to be destabilization-resistant and the conditions required to destabilize such memories remain poorly understood. Our lab has previously shown that exposure to salient novel contextual cues during memory reactivation can destabilize strongly encoded object location memories and that activity at muscarinic cholinergic receptors is critical for this effect. In the current study, we similarly targeted destabilization-resistant fear memories, hypothesizing that exposure to salient novelty at the time of reactivation would induce destabilization of strongly encoded fear memories in a muscarinic receptor-dependent manner. First, we show that contextual fear memories induced by 3 context-shock pairings readily destabilize upon memory reactivation, and that this destabilization is blocked by systemic (ip) administration of the muscarinic receptor antagonist scopolamine (0.3 mg/kg) in male rats. Following that, we confirm that this effect is dorsal hippocampus (dHPC)-dependent by targeting M1 receptors in the CA1 region with pirenzepine. Next, we show that more strongly encoded fear memories (induced with 5 context-shock pairings) resist destabilization. Consistent with our previous work, however, we report that salient novelty (a change in floor texture) presented during the reactivation session promotes destabilization of resistant contextual fear memories in a muscarinic receptor-dependent manner. Finally, the effect of salient novelty on memory destabilization was mimicked by stimulating muscarinic receptors with the selective M1 agonist CDD-0102A (ip, 0.3 mg/kg). These findings reveal further generalizability of our previous results implicating novel cues and M1 muscarinic signaling in promoting destabilization of resistant memories and suggest possible therapeutic options for disorders characterized by persistent, maladaptive fear memories such as PTSD and phobias.

SARS-CoV-2: analysis of the effects of mutations in non-structural proteins.

A worldwide pandemic that started in China in late 2019 was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded RNA virus belonging to the family Coronaviridae. Due to its structural variability and mutability, this virus continues to evolve and pose a major health threat around the world. Its characteristics, such as transmissibility, antigenicity, and resistance to drugs and vaccines, are continually altered through mutations. Examining mutational hotspots and their structural repercussions can thus aid in the development of more-effective vaccinations and treatment plans. In this study, we used full genome sequences of SARS-CoV-2 variants to predict structural changes in viral proteins. These sequences were obtained from the Global Initiative on Sharing Avian Influenza Data (GISAID), and a set of significant mutations were identified in each of the non-structural proteins (NSP1-16) and structural proteins, including the envelope, nucleocapsid, membrane, and spike proteins. The mutations were characterized as stabilizing or destabilizing based on their effect on protein dynamics and stability, and their impact on structure and function was evaluated. Among all of the proteins, NSP6 stands out as especially variable. The results of this study augment our understanding of how mutational events influence virus pathogenicity and evolution.

Misfolded protein oligomers induce an increase of intracellular Ca2+ causing an escalation of reactive oxidative species.

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid ß (Aß) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aß also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aß oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aß42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.

Application of Pluronics for Enhancing Aqueous Solubility of Lipophilic Microtubule Destabilizing Compounds on the Sea Urchin Embryo Model.

In screening, the dilution of DMSO stock solution of a lipophilic molecule with an assay medium often causes compound precipitation. To overcome the issue, the application of Pluronics as cosolvents was examined using a phenotypic sea urchin embryo assay that allows for the quick and facile evaluation of the antiproliferative effect together with systemic toxicity. Maximum tolerated concentration values for Pluronics L121, P123, and F127 were 1.4 µM, 8.6 µM, and 39.7 µM, respectively, and correlated directly with their hydrophilicity. Pluronics L121 and P123 suppressed cleavage and blastomeres retained the round shape, unlike hydrophilic Pluronic F127, which induced fertilization envelope creasing and embryo deformation that could be associated with the interaction of hydrophilic PEO units with mucopolysaccharides at the surface of sea urchin embryos. The toxicity of P123, but not of L121 and F127, was temperature-dependent and markedly increased at lower temperatures. CMC values obtained at different temperatures confirmed that the toxic effect of P123 was associated with both unimers and micelles, whereas F127 toxicity was related mainly to micelles. Evaluation using phenotypic sea urchin embryo assay revealed that potent microtubule destabilizers, namely albendazole, diarylisoxazole, and two chalcones, retained antimitotic activity after the dilution of their DMSO or 2-pyrrolidone stock solutions with 1.25% w/v Pluronic P123 or 5% w/v Pluronic F127. It was suggested that Pluronic P123 and Pluronic F127 could be used as cosolvents to improve the solubility of lipophilic molecules in aqueous medium.

The impact of glycerol monostearate's similarity to fats and fatty acid composition of fats on fat crystallization, destabilization, and texture properties of ice cream.

BACKGROUND: Fat significantly affects the properties of ice cream. Prior studies have investigated the correlation between fat crystallization, fat destabilization, and ice cream quality. However, the role of fatty acid composition, the similarity between fat and emulsifier in these characteristics, and their impact on final product quality remains unclear. RESULTS: To investigate the influence of the fatty acid composition of fats, as well as their similarity to glycerol monostearate (GMS), on fat crystallization and destabilization during the aging and freezing stages, ice creams were formulated using a combination of two types of fats (coconut oil and palm olein) in five different ratios. In oil phases, decreased saturation of fatty acids (from 93.38% to 46.69%) and increased similarity to GMS (from 11.96% to 46.01%) caused a reduction in the maximum solid fat content. Moreover, the rise in unsaturated long-chain fatty acids (from 34.61% to 99.57%) and similarity to GMS enhanced the formation of rare and coarse fat crystals, leading to a sparse crystalline network. This, in turn, reduced the crystallization rate and the stiffness of the fat in emulsions. Assuming consistent overrun across all ice creams, the enhanced interactions between fat globules in ice cream improved its hardness, melting properties, and shrinkage. CONCLUSION: The crystalline properties of fat in emulsions were influenced by oil phases, impacting fat destabilization and ultimately enhancing the quality of ice cream. The present study offers valuable insights for the optimization of fat and monoglyceride fatty acid ester selection, with the potential to improve ice cream quality. © 2023 Society of Chemical Industry.

Memory Destabilization and Reconsolidation Dynamically Regulate the PKMζ Maintenance Mechanism.

Useful memory must balance between stability and malleability. This puts effective memory storage at odds with plasticity processes, such as reconsolidation. What becomes of memory maintenance processes during synaptic plasticity is unknown. Here we examined the fate of the memory maintenance protein PKMζ during memory destabilization and reconsolidation in male rats. We found that NMDAR activation and proteasome activity induced a transient reduction in PKMζ protein following retrieval. During reconsolidation, new PKMζ was synthesized to re-store the memory. Failure to synthesize new PKMζ during reconsolidation impaired memory but uninterrupted PKMζ translation was not necessary for maintenance itself. Finally, NMDAR activation was necessary to render memories vulnerable to the amnesic effect of PKMζ-antisense. These findings outline a transient disruption and renewal of the PKMζ memory maintenance mechanism during plasticity. We argue that dynamic changes in PKMζ protein levels can serve as an exemplary model of the molecular changes underlying memory destabilization and reconsolidation.SIGNIFICANCE STATEMENT Maintenance of long-term memory relies on the persistent activity of PKMζ. However, after retrieval, memories can become transiently destabilized and must be reconsolidated within a few hours to persist. During this period of plasticity, what happens to maintenance processes, such as those involving PKMζ, is unknown. Here we describe dynamic changes to PKMζ expression during both destabilization and reconsolidation of auditory fear memory in the amygdala. We show that destabilization induces a NMDAR- and proteasome-dependent loss of synaptic PKMζ and that reconsolidation requires synthesis of new PKMζ. This work provides clear evidence that memory destabilization disrupts ongoing synaptic maintenance processes which are restored during reconsolidation.

Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

Systemic inhibition or global deletion of CaMKK2 protects against post-traumatic osteoarthritis.

OBJECTIVE: To investigate the role of Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA). METHODS: Destabilization of the medial meniscus (DMM) or sham surgeries were performed on 10-week-old male wild-type (WT) and Camkk2-/- mice. Half of the DMM-WT mice and all other cohorts (n = 6/group) received tri-weekly intraperitoneal (i.p.) injections of saline whereas the remaining DMM-WT mice (n = 6/group) received i.p. injections of the CaMKK2 inhibitor STO-609 (0.033 mg/kg body weight) thrice a week. Study was terminated at 8- or 12-weeks post-surgery, and knee joints processed for microcomputed tomography imaging followed by histology and immunohistochemistry. Primary articular chondrocytes were isolated from knee joints of 4-6-day-old WT and Camkk2-/- mice, and treated with 10 ng/ml interleukin-1ß (IL)-1ß for 24 or 48 h to investigate gene and protein expression. RESULTS: CaMKK2 levels and activity became elevated in articular chondrocytes following IL-1ß treatment or DMM surgery. Inhibition or absence of CaMKK2 protected against DMM-associated destruction of the cartilage, subchondral bone alterations and synovial inflammation. When challenged with IL-1ß, chondrocytes lacking CaMKK2 displayed attenuated inflammation, cartilage catabolism, and resistance to suppression of matrix synthesis. IL-1ß-treated CaMKK2-null chondrocytes displayed decreased IL-6 production, activation of signal transducer and activator of transcription 3 (Stat3) and matrix metalloproteinase 13 (MMP13), indicating a potential mechanism for the regulation of inflammatory responses in chondrocytes by CaMKK2. CONCLUSIONS: Our findings reveal a novel function for CaMKK2 in chondrocytes and highlight the potential for its inhibition as an innovative therapeutic strategy in the prevention of PTOA.

Dissociating the involvement of muscarinic and nicotinic cholinergic receptors in object memory destabilization and reconsolidation.

The content of long-term memory is neither fixed nor permanent. Reminder cues can destabilize consolidated memories, rendering them amenable to change before being reconsolidated. However, not all memories destabilize following reactivation. Characteristics of a memory, such as its age or strength, impose boundaries on destabilization. Previously, we demonstrated that presentation of salient novel information at the time of reactivation can readily destabilize resistant object memories in rats and this form of novelty-induced destabilization is dependent upon acetylcholine (ACh) activity at muscarinic receptors (mAChRs). In the present study, we sought to determine if this same mechanism for initiating destabilization of resistant object memories is present in mice and further expand our understanding of the mechanisms through which ACh modulates object memory destabilization by investigating the role of nicotinic receptors (nAChRs). We provide evidence that in mice mAChRs are necessary for destabilizing object memories that are readily destabilized and those that are resistant to destabilization. Conversely, nAChRs were found to be necessary only when memories are readily destabilized. We then investigated the role of both receptors in the reconsolidation of destabilized object memory traces and determined that nAChRs, but not mAChRs, are necessary for object memory reconsolidation. Together, these results suggest that nAChRs may play a more selective role in the re-storage of object memories following destabilization and that ACh acts through mAChRs to act as an override signal to initiate destabilization of resistant object memories following reactivation with novelty. These findings expand our current understanding of the role of ACh in the dynamic storage of long-term memory.

Coarse-Grained Molecular Dynamics Simulations of Paclitaxel-Loaded Polymeric Micelles.

A continuous manufacturing technology based on coaxial turbulent jet in coflow was previously developed to produce paclitaxel-loaded polymeric micelles. Herein, coarse-grained molecular dynamics (CG-MD) simulations were implemented to better understand the effect of the material attributes (i.e., the drug-polymer ratio and the ethanol concentration) and process parameters (i.e., temperature) on the self-assembly process of polymeric micelles as well as to provide molecular details on micelle instability. An all-atom (AA) poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) polymer model was developed as the reference for parameterizing a coarse-grained (CG) model, and the AA polymer model was further validated with experimental glass transition temperature (Tg). The model transferability was verified by comparing structural properties between the AA and CG models. The CG model was further validated with experimental data, including micelle particle size measurements and drug encapsulation efficiency. Furthermore, the encapsulation of paclitaxel into the polymeric micelles was included in the simulations, taking into consideration the interactions between the paclitaxel and the polymers. The results from various points of view demonstrated a strong dependence of the shape of the micelles on the drug encapsulation, with micelles transitioning from spherical to ellipsoidal structures with an increasing paclitaxel amount. Simulation data were also used to identify the critical aggregation number (i.e., the number of polymer and drug molecules required for transition from one shape to another). Improved micellar structural stability was found with a larger micellar size and less solvent accessibility. Lastly, an evaluation was performed on the micellar dissociation free energy using a steered molecular dynamics simulation over a range of temperatures and ethanol concentrations. These simulations revealed that at higher ethanol and temperature conditions, micelles become destabilized, resulting in greater paclitaxel release. The increased drug release was determined to originate from the solvation of the hydrophobic core, which promoted micellar swelling and an associated reduction in hydrophobic interactions, leading to a loosely packed micellar structure.