MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange.

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide-MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

The Clinical Research on Ginger (Zingiber officinale): Insights from ClinicalTrials.gov analysis.

Ginger (Zingiber officinale) has a rich history of traditional medicinal use and has attracted a global interest in its health benefits. This study aims to provide insights into the clinical research landscape on ginger, focusing on its pharmacological effects and studied health-related outcomes. The study design involves systematic analysis of data from clinical trials available on ClinicalTrials.gov and discussion of findings in the context of the existing scientific knowledge. A comprehensive analysis of clinical trials registered on ClinicalTrials.gov related to ginger was first conducted, and the scientific background related to specific ginger clinical research avenues was further evaluated through PubMed searches. A variety of trial designs were identified, including treatment, prevention, and supportive care objectives. A total of 188 studies were identified on ClinicalTrials.gov, of which 89 met the inclusion criteria. Among the 89 trials, treatment objectives were predominant (47.2%), and dietary supplements (40.4%) and drugs (27%) were the most prevalent intervention types. These trials covered various health outcomes, such as antiemetic activity, analgesic function, effects on health-related quality of life, blood pressure variation, energy expenditure, and reduction in xerostomia. This study analysis provides a comprehensive overview of the clinical trials landscape on ginger, focusing on its broad spectrum of potential health benefits. While individual trials show promising results, a significant gap in the available data with a low reporting rate of final results is identified, underscoring the need for further research to establish conclusive evidence of ginger's therapeutic potentials.

Design of a novel switchable antibody display system in Pichia pastoris.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomyces cerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichia pastoris (Komagataella phaffii). Establishment of surface display in P. pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments (DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P. pastoris formaldehyde dehydrogenase (FLD1) promoter, S. cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyces lactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S. cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: • First report of antibody display in P. pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

Controlling the Spatiotemporal Release of Nerve Growth Factor by Chitosan/Polycaprolactone Conduits for Use in Peripheral Nerve Regeneration.

Tubular polymeric structures have been recognized in the treatment of peripheral nerves as comparable to autologous grafting. The best therapeutic outcomes are obtained with conduits releasing therapeutic molecules. In this study, a new approach for the incorporation of biologically active agent-loaded microspheres into the structure of chitosan/polycaprolactone conduits was developed. The support of a polycaprolactone helix formed by 3D melt extrusion was coated with dopamine in order to adsorb nerve growth factor-loaded microspheres. The complex analysis of the influence of process factors on the coverage efficiency of polycaprolactone helix by nerve grow factor-loaded microspheres was analyzed. Thus, the PCL helix characterized with the highest adsorption of microspheres was subjected to nerve growth factor release studies, and finally incorporated into chitosan hydrogel deposit through the process of electrophoretic deposition. It was demonstrated by chemical and physical tests that the chitosan/polycaprolactone conduit meets the requirements imposed on peripheral nerve implants, particularly mimicking mechanical properties of surrounding soft tissue. Moreover, the conduit may support regrowing nerves for a prolonged period, as its structure and integrity persist upon incubation in lysozyme-contained PBS solution up to 28 days at body temperature. In vitro cytocompatibility toward mHippoE-18 embryonic hippocampal cells of the chitosan/polycaprolactone conduit was proven. Most importantly, the developed conduits stimulate axonal growth and support monocyte activation, the latter is advantageous especially at early stages of nerve regeneration. It was demonstrated that, through the described approach for controlling spatiotemporal release of nerve growth factors, these biocompatible structures adjusted to the specific peripheral nerve injury case can be manufactured.

Peripheral and central compensatory mechanisms for impaired vagus nerve function during peripheral immune activation.

BACKGROUND: Determining the etiology and possible treatment strategies for numerous diseases requires a comprehensive understanding of compensatory mechanisms in physiological systems. The vagus nerve acts as a key interface between the brain and the peripheral internal organs. We set out to identify mechanisms compensating for a lack of neuronal communication between the immune and the central nervous system (CNS) during infection. METHODS: We assessed biochemical and central neurotransmitter changes resulting from subdiaphragmatic vagotomy and whether they are modulated by intraperitoneal infection. We performed a series of subdiaphragmatic vagotomy or sham operations on male Wistar rats. Next, after full, 30-day recovery period, they were randomly assigned to receive an injection of Escherichia coli lipopolysaccharide or saline. Two hours later, animal were euthanized and we measured the plasma concentration of prostaglandin E2 (with HPLC-MS), interleukin-6 (ELISA), and corticosterone (RIA). We also had measured the concentration of monoaminergic neurotransmitters and their metabolites in the amygdala, brainstem, hippocampus, hypothalamus, motor cortex, periaqueductal gray, and prefrontal medial cortex using RP-HPLC-ED. A subset of the animals was evaluated in the elevated plus maze test immediately before euthanization. RESULTS: The lack of immunosensory signaling of the vagus nerve stimulated increased activity of discrete inflammatory marker signals, which we confirmed by quantifying biochemical changes in blood plasma. Behavioral results, although preliminary, support the observed biochemical alterations. Many of the neurotransmitter changes observed after vagotomy indicated that the vagus nerve influences the activity of many brain areas involved in control of immune response and sickness behavior. Our studies show that these changes are largely eliminated during experimental infection. CONCLUSIONS: Our results suggest that in vagotomized animals with blocked CNS, communication may transmit via a pathway independent of the vagus nerve to permit restoration of CNS activity for peripheral inflammation control.

Expression of hypoxia inducible factor 1α and 2α and its association with vitamin C level in thyroid lesions.

BACKGROUND: Cells adapt to hypoxia by transcriptional induction of genes that participate in regulation of angiogenesis, glucose metabolism and cell proliferation. The primary factors mediating cell response to low oxygen tension are hypoxia inducible factors (HIFs), oxygen-dependent transcription activators. The stability and activity of the α subunits of HIFs are controlled by hydroxylation reactions that require ascorbate as a cofactor. Therefore, deficiency of intracellular vitamin C could contribute to HIFs overactivation. In this study, we investigated whether vitamin C content of human thyroid lesions is associated with HIF-1α and HIF-2α protein levels. METHODS: Expression of HIF-1α and HIF-2α as well as vitamin C content was analyzed in thyroid lesions and cultured thyroid carcinoma cell lines (FTC-133 and 8305c) treated with hypoxia-mimetic agent (cobalt chloride) and ascorbic acid. The expression of HIFs and hypoxia-induced glucose transporters were determined by Western blots while quantitative real-time PCR (qRT-PCR) was performed to detect HIFs mRNA levels. Ascorbate and dehydroascorbate levels were measured by HPLC method. RESULTS: We found an inverse correlation between vitamin C level and HIF-1α but not HIF-2α expression in thyroid lesions. These results agree with our in vitro study showing that vitamin C induced a dose - dependent decrease of HIF-1α but not HIF-2α protein level in thyroid cancer cells FTC-133 and 8305C. The decreased HIF-1α expression was correlated with reduced expression of hypoxia-related glucose transporter 1 (GLUT1) in thyroid cancer cells. CONCLUSION: The results demonstrate that HIF-1α activation is associated with vitamin C content in thyroid lesions. Our study suggests that high tumor tissue ascorbate level could limit the expression of HIF-1α and its targets in thyroid lesions.

HLA-DMA polymorphisms differentially affect MHC class II peptide loading.

During the adaptive immune response, MHCII proteins display antigenic peptides on the cell surface of APCs for CD4(+) T cell surveillance. HLA-DM, a nonclassical MHCII protein, acts as a peptide exchange catalyst for MHCII, editing the peptide repertoire. Although they map to the same gene locus, MHCII proteins exhibit a high degree of polymorphism, whereas only low variability has been observed for HLA-DM. As HLA-DM activity directly favors immunodominant peptide presentation, polymorphisms in HLA-DM (DMA or DMB chain) might well be a contributing risk factor for autoimmunity and immune disorders. Our systematic comparison of DMA*0103/DMB*0101 (DMA-G155A and DMA-R184H) with DMA*0101/DMB*0101 in terms of catalyzed peptide exchange and dissociation, as well as direct interaction with several HLA-DR/peptide complexes, reveals an attenuated catalytic activity of DMA*0103/DMB*0101. The G155A substitution dominates the catalytic behavior of DMA*0103/DMB*0101 by decreasing peptide release velocity. Preloaded peptide-MHCII complexes exhibit ∼2-fold increase in half-life in the presence of DMA*0103/DMB*0101 when compared with DMA*0101/DMB*0101. We show that this effect leads to a greater persistence of autoimmunity-related Ags in the presence of high-affinity competitor peptide. Our study therefore reveals that HLA-DM polymorphic residues have a considerable impact on HLA-DM catalytic activity.

Susceptibility to HLA-DM protein is determined by a dynamic conformation of major histocompatibility complex class II molecule bound with peptide.

HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.

Effect of Glucose on GLUT1-Dependent Intracellular Ascorbate Accumulation and Viability of Thyroid Cancer Cells.

Enhanced glucose requirement of cancer cells is associated with an increased glucose transport across plasma membrane that is mediated by a family of facilitated glucose transporter proteins, named GLUTs. GLUT1 is the main transporter in thyroid cancer cells. Glucose is the principal physiological substrate of GLUT1; however, it is also capable of transporting of oxidized form of vitamin C [i.e., dehydroascorbic acid (DHAA) which inside the cells is reduced to ascorbic acid (AA)]. The objective of this study was to determine the effect of normo-, hypo-, and hyperglycemia conditions on GLUT1-dependent intracellular ascorbate accumulation and viability of thyroid cancer cells. GLUT1 seems to be the main DHAA transporter in thyroid cancer cells because its knockdown by RNAi reduced DHAA accumulation by more than 80%. The results showed that in thyroid cancer cells high glucose inhibits both transport of AA and DHAA. Inhibition of vitamin C transport by glucose had a cytotoxic effect on the cells. However, stabilization of vitamin C in one of 2 forms (i.e., AA or DHAA) abolished this effect. These results suggest that cytotoxic effect is rather associated with extracellular accumulation of vitamin C and changes of its oxidation state than with intracellular level of ascorbate.

Peptide-polymer ligands for a tandem WW-domain, an adaptive multivalent protein-protein interaction: lessons on the thermodynamic fitness of flexible ligands.

Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3-9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide-polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide-polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3-2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide-polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

Social stress increases expression of hemoglobin genes in mouse prefrontal cortex.

BACKGROUND: In order to better understand the effects of social stress on the prefrontal cortex, we investigated gene expression in mice subjected to acute and repeated social encounters of different duration using microarrays. RESULTS: The most important finding was identification of hemoglobin genes (Hbb-b1, Hbb-b2, Hba-a1, Hba-a2, Beta-S) as potential markers of chronic social stress in mice. Expression of these genes was progressively increased in animals subjected to 8 and 13 days of repeated stress and was correlated with altered expression of Mgp (Mglap), Fbln1, 1500015O10Rik (Ecrg4), SLC16A10, and Mndal. Chronic stress increased also expression of Timp1 and Ppbp that are involved in reaction to vascular injury. Acute stress did not affect expression of hemoglobin genes but it altered expression of Fam107a (Drr1) and Agxt2l1 (Etnppl) that have been implicated in psychiatric diseases. CONCLUSIONS: The observed up-regulation of genes associated with vascular system and brain injury suggests that stressful social encounters may affect brain function through the stress-induced dysfunction of the vascular system.

Histone H3 posttranslational modified enzymes defined neutrophil plasticity and their vulnerability to IL-10 in the course of the inflammation.

BACKGROUND: Neutrophils are a heterogeneous population capable of antimicrobial functions associated with pre-activation/activation and tissue regeneration. The specific polarisation of immune cells is mediated by the modification of 'chromatin landscapes', which enables differentiated access and activity of regulatory elements that guarantee their plasticity during inflammation No specific pattern within histone posttranslational modifications (PTMs) controlling this plasticity has been identified. METHODS: Using the in vitro model of inflammation, reflecting different states of neutrophils from resting, pre-activated cells to activated and reducing tissue regeneration, we have analysed 11 different histone posttranslational modifications (PTMs), PTM enzymes associated with remodelling neutrophil chromatin, and H3K4me3 ChIP-Seq Gene Ontology analysis focusing on the processes related to histone PTMs. These findings were verified by extrapolation to adequate clinical status, using neutrophils derived from the patients with sepsis (systemic septic inflammation with LPS-stimulated neutrophils), neuromyelitis optical spectrum disorders (aseptic inflammation with pre-activated neutrophils) and periodontitis (local self-limiting septic inflammation with IL-10-positive neutrophils). RESULTS: Physiological activation of neutrophils comprises a pre-activation characterised by histone H3K27ac and H3K4me1, which position enhancers; direct LPS exposure is induced explicitly by H3K4me3 which marked Transcription Start Site (TSS) regions and low-level of H3K9me3, H3K79me2 and H3K27me3 which, in turn, marked repressed genes. Contrary to antimicrobial action, IL-10 positively induced levels of H3S10p and negatively H3K9me3, which characterised processes related to the activation of genes within heterochromatin mediated by CHD1 and H3K9me3 specific demethylase JMJD2A. IL-10 protects changes within histone PTMs induced by TNF or LPS that affected H3K4me3-specific methyltransferase SETD1A and MLL1. Neutrophils previously exposed to inflammatory factors become unvulnerable to IL-10 because previous LPS stimulation interrupts TSS regions marked by H3K4me3 of CHD1 and JMJD2A genes. Therefore, LPS-activated neutrophils are disabled to induce CHD1/JMJD2A enzymes by IL-10, making this process irreversible. Because transcription of JMJD2A and CHD1 also depends on TSS positioning by H3K4me3, neutrophils before LPS stimulation become insensitive to IL-10. CONCLUSION: Neutrophils, once pre-activated by TNF or directly stimulated by LPS, become insensitive to the anti-inflammatory effects of IL-10, and vice versa; IL-10 protects neutrophils against these proinflammatory stimuli. This phenomenon is responsible for disturbing the natural process of resolving inflammation and tissue regeneration.

Stress susceptibility-specific phenotype associated with different hippocampal transcriptomic responses to chronic tricyclic antidepressant treatment in mice.

BACKGROUND: The effects of chronic treatment with tricyclic antidepressant (desipramine, DMI) on the hippocampal transcriptome in mice displaying high and low swim stress-induced analgesia (HA and LA lines) were studied. These mice displayed different depression-like behavioral responses to DMI: stress-sensitive HA animals responded to DMI, while LA animals did not. RESULTS: To investigate the effects of DMI treatment on gene expression profiling, whole-genome Illumina Expression BeadChip arrays and qPCR were used. Total RNA isolated from hippocampi was used. Expression profiling was then performed and data were analyzed bioinformatically to assess the influence of stress susceptibility-specific phenotypes on hippocampal transcriptomic responses to chronic DMI. DMI treatment affected the expression of 71 genes in HA mice and 41 genes in LA mice. We observed the upregulation of Igf2 and the genes involved in neurogenesis (HA: Sema3f, Ntng1, Gbx2, Efna5, and Rora; LA: Otx2, Rarb, and Drd1a) in both mouse lines. In HA mice, we observed the upregulation of genes involved in neurotransmitter transport, the termination of GABA and glycine activity (Slc6a11, Slc6a9), glutamate uptake (Slc17a6), and the downregulation of neuropeptide Y (Npy) and corticotropin releasing hormone-binding protein (Crhbp). In LA mice, we also observed the upregulation of other genes involved in neuroprotection (Ttr, Igfbp2, Prlr) and the downregulation of genes involved in calcium signaling and ion binding (Adcy1, Cckbr, Myl4, Slu7, Scrp1, Zfp330). CONCLUSIONS: Several antidepressant treatment responses are similar in individuals with different sensitivities to stress, including the upregulation of Igf2 and the genes involved in neurogenesis. However, the findings also reveal that many responses to antidepressant treatments, involving the action of individual genes engaged in neurogenesis, neurotransmitter transport and neuroprotection, depend on constitutive hippocampal transcriptomic profiles and might be genotype dependent. The results suggest that, when and if this becomes feasible, antidepressant treatment should take into consideration individual sensitivity to stress.

Sex-dependent neurotransmitter level changes in brains of Toxoplasma gondii infected mice.

The protozoan parasite Toxoplasma gondii has the ability to alter intermediate host behavior, most impressively the natural aversion to cat scent, to favor the predation by the definitive host. However, the underlying mechanism of the observed phenomenon still remains unknown. Since changes in the neurotransmitter level are postulated as a possible contributing factor, the aim of this work was to assess the monoamine systems activity in specified brain regions involved in the natural defense behaviors, emotion evaluation, and motor and sensory stimuli integration in experimentally T. gondii infected mice compared to uninfected controls. Taking into account the natural differences between genders, the experiments were carried out on both male and female mice. Our results revealed statistically significant changes in all tested monoamine systems with regard to both gender and time after T. gondii invasion. Acute toxoplasmosis was accompanied by a decrease in noradrenergic system activity in females and its slight increase in some brain areas of males. Acute invasion also induced a rise in serotonin system activity, mostly in males. The most striking observation was an increase in the dopamine release noted in acutely infected males. We discuss our results in terms of their possible contribution to T. gondii-induced intermediate host behavior alterations and parasite transmission and with regard to postulated relationship between T. gondii seroprevalence and occurrence of certain disorders such as schizophrenia in humans.

Effect of selective cyclooxygenase inhibitors on animal behaviour and monoaminergic systems of the rat brain.

The long-term effects of cyclooxygenase 1 and 2 (COX-1/2) inhibitors are usually tested in terms of the periphery of the organism. Therefore, we studied the effects of SC560 (selective COX-1 inhibitor) and celecoxib (selective COX-2 inhibitor) on the activity of brain monoaminergic systems and animal behaviour. Additionally, we tested the effect of these inhibitors during inflammation. We have observed that long-term administration of celecoxib reduces the activity of the noradrenergic system, increases the activity of dopaminergic and serotonergic systems, increases locomotor activity, and enhances the exploratory behaviour of rats. Administration of SC560 also increases the activity of dopaminergic and serotonergic systems but reduces locomotor activity and impairs the exploratory behaviour of rats. The mechanism responsible for decreased activity of the noradrenergic system may be related to the weakening of activity of the positive feedback loop between the paraventricular nucleus and coeruleus locus. We suggest that the effect of used inhibitors on the dopaminergic system is associated with a possible increase in anandamide concentration and its effect on dopamine reuptake in synaptic clefts. It also appears that cyclooxygenase peroxidase activity may play a role in this process. In turn, changes in the activity of the serotonergic system may be related to the activity of indoleamine-2,3-dioxygenase, which decreases because of the decreased concentration of pro-inflammatory compounds. We believe that behavioural changes induced by COX inhibitors are the result of the modified activity of monoaminergic CNS systems in the brainstem, hypothalamus, and medial prefrontal cortex.

Bilateral subdiaphragmatic vagotomy modulates the peripheral met­enkephalin and striatal monoamine responses to peripheral inflammation in rat.

In the central nervous system, long­term effects of a vagotomy include disturbance of monoaminergic activity of the limbic system. Since low vagal activity is observed in major depression and autism spectrum disorder, the study aimed to determine whether animals fully recovered after subdiaphragmatic vagotomy demonstrates neurochemical indicators of altered well­being and social component of sickness behavior. Bilateral vagotomy or sham surgery was performed in adult rats. After one month of recovery, rats were challenged with lipopolysaccharide or vehicle to determine the role of central signaling upon sickness. Striatal monoamines and met­enkephalin concentrations were evaluated using HPLC and RIA methods. We also defined a concentration of immune­derived plasma met­enkephalin to establish a long­term effect of vagotomy on peripheral analgesic mechanisms. The data indicate that 30 days after vagotomy procedure, striatal dopaminergic, serotoninergic, and enkephalinergic neurochemistry was altered, both under physiological and inflammatory conditions. Vagotomy prevented inflammation­induced increases of plasma met­enkephalin - an opioid analgesic. Our data suggest that in a long perspective, vagotomized rats may be more sensitive to pain and social stimuli during peripheral inflammation.

Class I HDAC Inhibition Leads to a Downregulation of FANCD2 and RAD51, and the Eradication of Glioblastoma Cells.

HDAC inhibitors (HDACi) hold great potential as anticancer therapies due to their ability to regulate the acetylation of both histone and non-histone proteins, which is frequently disrupted in cancer and contributes to the development and advancement of the disease. Additionally, HDACi have been shown to enhance the cytotoxic effects of DNA-damaging agents such as radiation and cisplatin. In this study, we found that histone deacetylase inhibits valproic acid (VPA), synergized with PARP1 inhibitor (PARPi), talazoparib (BMN-673), and alkylating agent, and temozolomide (TMZ) to induce DNA damage and reduce glioblastoma multiforme. At the molecular level, VPA leads to a downregulation of FANCD2 and RAD51, and the eradication of glioblastoma cells. The results of this study indicate that combining HDACi with PARPi could potentially enhance the treatment of glioblastoma, the most aggressive type of cancer that originates in the brain.

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Behavioral changes in mice caused by Toxoplasma gondii invasion of brain.

Toxoplasma gondii, a protozoan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite undergoes sexual reproduction resulting in genetic variability only in the intestine of the definitive host (a member of the cat family). The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the number of parasite cysts formed in the hippocampus and amygdala of experimentally infected mice as these regions are involved in defense behaviors control and emotion processing, and to assess the influence of the infection on mice behavior. The obtained results revealed the presence of parasite cysts both in the hippocampus and the amygdala of infected mice; however, no clear region-dependent distribution was observed. Furthermore, infected mice showed significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central, more exposed part of the OF arena and engaged in less grooming behavior compared to uninfected controls.

A switchable secrete-and-capture system enables efficient selection of Pichia pastoris clones producing high yields of Fab fragments.

Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened. In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and-capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS). Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones. Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha-agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and-capture system offers a switchable scFv display. Efficient plasmid clearance upon removal of selective pressure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (factor in the range of ∼21-49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone. Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of ∼2-3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.