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Neurofilament light chain protein (NfL) levels reflect neuronal damage in several neurological diseases and have been proposed as a possible biomarker. Plasma extracellular vesicles (EVs) could play an important role as mediators of the inflammatory changes associated with inducing minimal hepatic encephalopathy (MHE) in cirrhotic patients. This study investigated the association of NfL levels in plasma and EVs with the presence of MHE in cirrhotic patients, and with responses to rifaximin treatment. The NfL levels in plasma and EVs were assessed in 71 patients with liver cirrhosis (40 with MHE and 31 without MHE) and 26 controls. A total of 31 patients with MHE received rifaximin treatment. We examined changes in NfL levels in plasma and EVs before and after 6 months of rifaximin treatment. The NfL measures were correlated with cognitive alterations and plasma inflammatory cytokines. MHE patients showed increased plasma levels of NfL, which were reverted after rifaximin treatment in patients who responded to treatment. The NfL content in EVs also showed a reversal pattern in MHE patients treated with rifaximin. In multivariable analyses, NfL levels were independently associated with the presence of MHE. We also showed that patients with high levels of both ammonia and fractalkine had significantly higher NfL levels than patients with low levels of least one of these parameters. Rifaximin treatment in MHE patients showed promising results in improving axonal damage, suggesting that rifaximin may have therapeutic benefits against disease progression in MHE.

Patients with nonalcoholic fatty liver disease (NAFLD) may show mild cognitive impairment (MCI). The mechanisms involved remain unclear. The plasma concentrations of several cytokines and chemokines were measured in 71 NAFLD patients (20 with and 51 without MCI) and 61 controls. Characterization and activation of leukocyte populations and CD4 + sub-populations were carried out and analyzed by flow cytometry. We analyzed the cytokines released from CD4 + cell cultures and the mRNA expression of transcription factors and receptors in peripheral blood mononuclear cells. The appearance of MCI in NAFLD patients was associated with increased activation of CD4 + T lymphocytes, mainly of the Th17 subtype, increased plasma levels of pro-inflammatory and anti-inflammatory cytokines such as IL-17A, IL-23, IL-21, IL-22, IL-6, INF-γ, and IL-13, and higher expression of the CCR2 receptor. Constitutive expression of IL-17 was found in cultures of CD4 + cells from MCI patients, reflecting Th17 activation. High IL-13 plasma levels were predictive of MCI and could reflect a compensatory anti-inflammatory response to the increased expression of pro-inflammatory cytokines. This study identified some specific alterations of the immune system associated with the appearance of neurological alterations in MCI patients with NAFLD that could be the basis to improve and restore cognitive functions and quality of life in these patients.

The increase of vascular arginase activity during aging causes endothelial dysfunction. This enzyme competes with the endothelial nitric oxide synthase (eNOS) for L-arginine substrate. Our hypothesis is that glucose 6-P dehydrogenase (G6PD) overexpression could improve the endothelial function modulating the arginase pathway in aorta from mice. For this study, three groups of male mice were used: young wild type (WT) (6-9 months), old WT (21-22 months) and old G6PD-Tg (21-22 months) mice. Vascular reactivity results showed a reduced acetylcholine-dependent relaxation in the old WT but not old G6PD-Tg group. Endothelial dysfunction was reverted by nor-NOHA, an arginase inhibitor. Mice overexpressing G6PD underexpressed arginase II and also displayed a lower activity of this enzyme. Moreover, histological analyses demonstrated that age causes a thickness of aortic walls, but this did not occur in G6PD-Tg mice. We conclude that the overexpressing G6PD mouse is a model to improve vascular health via the arginase pathway.

Meningioma is the most common intracranial tumor in adulthood. With a clear female predominance and a recurrence rate that reaches 20%, it is, despite being considered a benign tumor, a pathology that greatly compromises post-diagnosis quality of life. Its prone to recur or progress to a higher degree is difficult to predict in the absence of obvious histological criteria. This project aims to develop an automatic methodology to aid in the diagnosis of meningiomas that is objective and easily reproducible. The methodology is based on histopathological image analysis using artificial intelligence and machine learning algorithms. It includes a semi-automatic process of identification and cleaning of the scanned samples, an automatic detection of the nuclei of each image and, finally, the parameterization of the samples. The obtained data together with the clinical information will be analyzed using statistical methods in order to provide a methodology to support clinical diagnosis and decision-making in patient management. The result is the development of an effective methodology that generates a set of data associated with morphological parameters with different trends according to the pathological groups studied. A tool has been developed that allows an effective semiautomatic analysis of the images to evaluate these parameters in an objective and reproducible way, helping in clinical decision-making and facilitating to undertake projects with large sample series. Clinical Relevance- The main contribution of this project is in the field of neuropathology, for the diagnosis of meningiomas, the most common brain tumor. The present project provides an objective and quantifiable prognosis methodology for the meningiomas, offering a more precise monitoring of the treatment applied to the patient, resulting in a better prognosis and better quality of life.

Desmoplastic infantile astrocytoma (DIA) is rare, cystic and solid tumor of infants usually found in superficial cerebral hemispheres. Although DIA is usually benign, uncommon cases bearing malignant histological and aggressive clinical features have been described in the literature. We report a newborn patient who was diagnosed with a DIA and died postresection. Pathologic examination revealed that the main part of the tumor had benign features, but the internal region showed areas with a more aggressive appearance, with higher-proliferative cells, anaplastic GFAP positive cells with cellular polymorphism, necrosis foci, vascular hyperplasia with endothelial proliferation and microtrombosis. Genetic study, performed in both regions of the tumor, showed a BRAF V600E mutation and a homozygous deletion in PTEN, without changes in other relevant genes like EGFR, CDKN2A, TP53, NFKBIA, CDK4, MDM2 and PDGFRA. Although PTEN homozygous deletions are described in gliomas, the present case constitutes the first report of a PTEN mutation in a DIA, and this genetic feature may be related to the malignant behavior of a usually benign tumor. These genetic findings may point at the need of further and deeper genetic characterization of DIAs, in order to better understand the biology of this tumor and to obtain new prognostic approaches, a better clinical management and targeted therapies, especially in malignant cases of DIA., (© 2022. The Author(s).)

Patients with cirrhosis may show minimal hepatic encephalopathy (MHE), for which rifaximin is effective. Metabolic syndrome may be associated with cognitive impairment. Our aims were to evaluate the influence of metabolic syndrome features on response to rifaximin for neurological and inflammatory alterations in MHE. A prospective cohort study was conducted in 63 cirrhotic patients and 30 controls from two tertiary centres recruited between 2015 and 2019. Metabolic syndrome was defined according to the Adult Treatment Panel-III. Patients were classified into 31 without and 32 with MHE according to the Psychometric Hepatic Encephalopathy Score (PHES). All participants performed specific psychometric tests, and inflammatory parameters were studied. Patients with MHE received rifaximin (400 mg/8 h). Response was evaluated by PHES at 3 and 6 months. Response according to metabolic syndrome manifestations was compared. The response rate was 66%. Older age (p = 0.012) and all metabolic syndrome diseases (p < 0.05) were associated with non-response, plus an increase in risk as the number of manifestations rose (p < 0.001). Patients with metabolic manifestations exhibited worse processing speed (p = 0.011), working memory (p = 0.005), visual coordination (p = 0.013) and lower proportion of activated CD4 + lymphocytes (p = 0.039) at baseline, as well as worse concentration (p = 0.030), bimanual coordination (p = 0.004) and higher levels of intermediate monocytes (p = 0.026), CX3CL1 (p < 0.05), IL-17 (p = 0.022), AHR (p = 0.010) and IgG (p < 0.05) at 3 and/or 6 months of rifaximin. Patients with clinical signs of metabolic syndrome have poor response to rifaximin for MHE, with a higher proportion of neurological alterations associated with a pro-inflammatory environment., (© 2022. The Author(s).)

Patients with nonalcoholic fatty liver disease (NAFLD) may show mild cognitive impairment (MCI). The neurological functions affected remain unclear. The aims were to: (1) Characterize the neuropsychological alterations in NAFLD patients; (2) assess the prevalence of impairment of neurological functions evaluated; (3) develop a new score for sensitive and rapid MCI detection in NAFLD; (4) assess differences in MCI features between patients with nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH); and (5) compare neuropsychological alterations in NAFLD patients with cirrhotic patients with MCI. Fifty-nine NAFLD patients and 53 controls performed psychometric tests assessing different neurological functions: PHES (Psychometric Hepatic Encephalopathy Score) battery, d2, Stroop, Oral SDMT (Symbol Digit Modalities Test), Digit Span, number-letter test, and bimanual and visual-motor coordination tests. NAFLD patients show impairment in attention, mental concentration, psychomotor speed, cognitive flexibility, inhibitory mental control, and working memory. We developed a new, rapid, and sensitive score based on the most affected parameters in NAFLD patients, unveiling that 32% of NAFLD show MCI. Prevalence was similar in NAFL (36%) or NASH (27%) patients, but lower in NAFLD than in cirrhosis (65%). MCI prevalence is significant in NAFLD patients. Psychometric testing is warranted in these patients to unveil MCI and take appropriate measures to reverse and prevent its progression.

Cirrhotic patients may experience alterations in the peripheral nervous system and in somatosensory perception. Impairment of the somatosensory system could contribute to cognitive and motor alterations characteristic of minimal hepatic encephalopathy (MHE), which affects up to 40% of cirrhotic patients. We assessed the relationship between MHE and alterations in thermal, vibration, and/or heat pain sensitivity in 58 cirrhotic patients (38 without and 20 with MHE according to Psychometric Hepatic Encephalopathy Score) and 39 controls. All participants underwent attention and coordination tests, a nerve conduction study, autonomic function testing, and evaluation of sensory thresholds (vibration, cooling, and heat pain detection) by electromyography and quantitative sensory testing. The detection thresholds for cold and heat pain on the foot were higher in patients with, than those without MHE. This hyposensitivity was correlated with attention deficits. Reaction times in the foot were longer in patients with, than without MHE. Patients with normal sural nerve amplitude showed altered thermal sensitivity and autonomic function, with stronger alterations in patients with, than in those without MHE. MHE patients show a general decrease in cognitive and sensory abilities. Small fibers of the autonomic nervous system and thermal sensitivity are altered early on in MHE, before large sensory fibers. Quantitative sensory testing could be used as a marker of MHE.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and GBM stem cells (GSC) may be responsible for its recurrence and therapeutic resistance. Toll-like receptors (TLRs), which recognize multiple ligands (endogenous and pathogen-associated) and trigger the immune response of mature immune cells, are also expressed by hematopoietic stem and progenitor cells, where their activation results in the differentiation of these cells into myeloid cells. Since TLR expression has been recently described in neural cells, including neural stem cells, we studied TLR expression by GSCs and the effect of stimulation by TLR ligands on promoting GSC differentiation into mature GBM cells. First, our results showed heterogeneous TLR expression by GBM cells from human tumors and, for the first time, by human GSCs defined by their CD133 + and CD44 + phenotypes. Next, the effect of TLR ligands was studied in in vitro cell cultures of neurospheres and CD44 + cells obtained from two GBM cell lines (U-87 and U-118). The expression of GSC markers diminished in the presence of Pam 3 CSK 4 or LPS (TLR2 and TLR4 ligands, respectively), thus indicating TLR-dependent differentiation. Interestingly, simultaneous treatment with Pam 3 CSK 4 plus temozolomide (TMZ), the reference drug in GBM treatment, significantly increased cell death compared to the effect of the ligand alone, which showed no toxicity, or TMZ alone. These results suggest a synergistic effect between Pam 3 CSK 4 and TMZ based on the induction of TLR-dependent GSC differentiation towards mature GBM cells, which exhibited increased sensitivity to chemotherapy, and provide new perspectives in GBM therapy.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours 1,2 . The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity 3-5 . Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRAS G12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C , which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRAS G12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRAS G12C with submicromolar inhibition of downstream signaling in a KRAS G12C -specific manner., Competing Interests: The authors declare no competing financial interest.

The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desalting step. Accurate protein masses are measured (±0.5 amu of the theoretical molecular weight; measured precision of ±0.02 amu). The fraction of protein reacted with an electrophilic compound is determined relative to an unmodified protein control. A key element of the workflow is the automated identification and quantitation of the expected masses of covalent protein-inhibitor adducts using a custom routine that obviates the need to manually inspect each individual spectrum. Parallel screens were performed on a library of approximately 1000 acrylamide containing compounds (different structures and reactivities) using the solid-phase extraction mass spectrometry based assay and a fluorescence based thiol-reactive probe assay enabling comparison of false positives and false negatives between these orthogonal screening approaches., (© 2015 Society for Laboratory Automation and Screening.)

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

Based on lead compound 1, which was discovered from a high-throughput screen, a series of PI3Kα/mTOR inhibitors were evaluated that contained an imidazo[1,2-a]pyridine as a core replacement for the benzimidazole contained in 1. By exploring various ring systems that occupy the affinity pocket, two fragments containing a methoxypyridine were identified that gave <100 nM potency toward PI3Kα in enzyme and cellular assays with moderate stability in rat and human liver microsomes. With the two methoxypyridine groups selected to occupy the affinity pocket, analogs were prepared with various fragments intended to occupy the ribose pocket of PI3Kα and mTOR. From these analogs, tertiary alcohol 18 was chosen for in vivo pharmacodynamic evaluation based on its potency in the PI3Kα cellular assay, microsomal stability, and in vivo pharmacokinetic properties. In a mouse liver pharmacodynamic assay, compound 18 showed 56% inhibition of HFG-induced AKT (Ser473) phosphorylation at a 30 mg/kg dose., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Rhabdomyosarcomas (RMS) are soft tissue malignant tumors of childhood and adolescents. The mechanisms underlying their aggressiveness are still poorly understood. Chemokines are chemotactic proteins involved in pathological processes that have been intensely studied in several types of cancers because of their influence in migration, angiogenesis, or metastases. We analyzed the expression of the chemokine receptors CXCR3, CXCR4 and CXCR7 and their ligands CXCL9, CXCL10, CXCL11 and CXCL12, in 15 RMS samples derived from nine patients. Expression was measured in tumors and primary cultures of RMS by Real-Time Polymerase Chain Reaction, immunostaining and flow cytometry. Our results show that these receptors are widely expressed in RMS. A significant difference between CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR7 expression ratios was found in alveolar versus embryonal RMS and similarly between CXCL12/CXCR4 and CXCL11/CXCR3 ratios in primary versus recurrent tumors. These findings suggest a possible association between the interrelation of chemokine/chemokine-receptor and an aggressive biological behavior in RMS.

2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Replacement of the piperazine sulfonamide portion of the PI3Kα inhibitor AMG 511 (1) with a range of aliphatic alcohols led to the identification of a truncated gem-dimethylbenzylic alcohol analog, 2-(5-(4-amino-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)propan-2-ol (7). This compound possessed good in vitro efficacy and pharmacokinetic parameters and demonstrated an EC50 of 239 ng/mL in a mouse liver pharmacodynamic model measuring the inhibition of hepatocyte growth factor (HGF)-induced Akt Ser473 phosphorylation in CD1 nude mice 6 h post-oral dosing., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Chemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival., Methods: In this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed., Results: Our results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface., Conclusions: Coexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

Background: Producing recombinant plant proteins expressed in Escherichia coli produce in high yields and in a soluble and functional form can be difficult. Under overexpression conditions, proteins frequently accumulate as insoluble aggregates (inclusion bodies) within the producing bacteria. We evaluated how the initial culture density, temperature and duration of the expression stage affect the production of some eukaryotic enzymes in E. coli., Findings: A high yield of active soluble proteins was obtained by combining early-log phase cultures and low temperatures for protein induction. When IPTG was added at OD600 = 0.1 and cultures were maintained at 4°C for 48-72 h, the soluble protein yield was 3 fold higher than that obtained in the mid-log phase (OD600 = 0.6). Besides, the target protein expression increased and the endogenous bacterial proteins reduced, thus making the protein purification process easier and more efficient., Conclusions: The protocol can be widely applied to proteins with a heterologous expression which was limited by loss of activity at high temperatures or by low soluble recombinant protein yield.