HuR controls glutaminase RNA metabolism.

Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.

Pediatric-Like Brain Tumors in Adults.

Pediatric brain tumors are different to those found in adults in pathological type, anatomical site, molecular signature, and probable tumor drivers. Although these tumors usually occur in childhood, they also rarely present in adult patients, either as a de novo diagnosis or as a delayed recurrence of a pediatric tumor in the setting of a patient that has transitioned into adult services.Due to the rarity of pediatric-like tumors in adults, the literature on these tumor types in adults is often limited to small case series, and treatment decisions are often based on the management plans taken from pediatric studies. However, the biology of these tumors is often different from the same tumors found in children. Likewise, adult patients are often unable to tolerate the side effects of the aggressive treatments used in children-for which there is little or no evidence of efficacy in adults. In this chapter, we review the literature and summarize the clinical, pathological, molecular profile, and response to treatment for the following pediatric tumor types-medulloblastoma, ependymoma, craniopharyngioma, pilocytic astrocytoma, subependymal giant cell astrocytoma, germ cell tumors, choroid plexus tumors, midline glioma, and pleomorphic xanthoastrocytoma-with emphasis on the differences to the adult population.

Toward the "Perfect" Shunt: Historical Vignette, Current Efforts, and Future Directions.

As a concept, drainage of excess fluid volume in the cranium has been around for more than 1000 years. Starting with the original decompression-trepanation of Abulcasis to modern programmable shunt systems, to other nonshunt-based treatments such as endoscopic third ventriculostomy and choroid plexus cauterization, we have come far as a field. However, there are still fundamental limitations that shunts have yet to overcome: namely posture-induced over- and underdrainage, the continual need for valve opening pressure especially in pediatric cases, and the failure to reinstall physiologic intracranial pressure dynamics. However, there are groups worldwide, in the clinic, in industry, and in academia, that are trying to ameliorate the current state of the technology within hydrocephalus treatment. This chapter aims to provide a historical overview of hydrocephalus, current challenges in shunt design, what members of the community have done and continue to do to address these challenges, and finally, a definition of the "perfect" shunt is provided and how the authors are working toward it.

Architecture and regulation of filamentous human cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

The influence of subclinical active inflammation on IFX pharmacokinetic modeling and disease progression assessment: findings from a prospective real-world study in inflammatory bowel disease patients.

BACKGROUND AND AIMS: Effective management of inflammatory bowel disease (IBD) relies on a comprehensive understanding of infliximab (IFX) pharmacokinetics (PK). This study's primary goal was to develop a robust PK model, identifying key covariates influencing IFX clearance (CL), while concurrently evaluating the risk of disease progression during the maintenance phase of IBD treatment. METHODS: The multicenter, prospective, real-world DIRECT study was conducted in several care centers, which included 369 IBD patients in the maintenance phase of IFX therapy. A two-compartment population PK model was used to determine IFX CL and covariates. Logistic and Cox regressions were applied to elucidate the associations between disease progression and covariates embedded in the PK model. RESULTS: The PK model included the contributions of weight, albumin, antidrug antibody (ADA), and fecal calprotectin (FC). On average, higher ADA, FC concentration and weight, and lower albumin concentration resulted in higher IFX CL. In the multivariate regression analyses, FC levels influenced the odds of disease progression in all its different definitions, when adjusted for several confounding factors. Additionally, alongside FC, both IFX and CL demonstrated a significant impact on the temporal aspect of disease progression. CONCLUSION: In this 2-year real-world study, readily available clinical covariates, notably FC, significantly impacted IFX availability in IBD patients. We demonstrated that subclinical active inflammation, as mirrored by FC or CRP, substantially influenced IFX clearance. Importantly, FC emerged as a pivotal determinant, not only of IFX pharmacokinetics but also of disease progression. These findings underscore the need to integrate FC into forthcoming IFX pharmacokinetic models, amplifying its clinical significance.

HER2 aptamer-conjugated iron oxide nanoparticles with PDMAEMA-b-PMPC coating for breast cancer cell identification.

Aim: To synthesize HER2 aptamer-conjugated iron oxide nanoparticles with a coating of poly(2-(dimethylamino) ethyl methacrylate)-poly(2-methacryloyloxyethylphosphorylcholine) block copolymer (IONPPPs). Methods: Characterization covered molecular structure, chemical composition, thermal stability, magnetic characteristics, aptamer interaction, crystalline nature and microscopic features. Subsequent investigations focused on IONPPPs for in vitro cancer cell identification. Results: Results demonstrated high biocompatibility of the diblock copolymer with no significant toxicity up to 150 µg/ml. The facile coating process yielded the IONPP complex, featuring a 13.27 nm metal core and a 3.10 nm polymer coating. Functionalized with a HER2-targeting DNA aptamer, IONPPP enhanced recognition in HER2-amplified SKBR3 cells via magnetization separation. Conclusion: These findings underscore IONPPP's potential in cancer research and clinical applications, showcasing diagnostic efficacy and HER2 protein targeting in a proof-of-concept approach.

Thiopurines have no impact on outcomes of Crohn's disease patients beyond 12 months of maintenance treatment with infliximab.

BACKGROUND: The emergence of new treatments the inflammatory bowel diseases (IBD) raised questions regarding the role of older agents, namely thiopurines. AIMS: To clarify the benefits of combination treatment with thiopurines on Crohn's disease (CD) patients in the maintenance phase of infliximab. METHODS: In this analysis of the 2-year prospective multicentric DIRECT study, patients were assessed in terms of clinical activity, faecal calprotectin (FC), C-reactive protein (CRP), and infliximab pharmacokinetics. A composite outcome based on clinical- and drug-related items was used to define treatment failure. RESULTS: The study included 172 patients; of these, 35.5 % were treated with combination treatment. Overall, 18 % of patients achieved the composite outcome, without statistically significant differences between patients on monotherapy and on combination treatment (21.6% vs 11.5 %, p = 0.098). Median CRP, FC, and infliximab pharmacokinetic parameters were similar in both groups. However, in the sub-analysis by infliximab treatment duration, in patients treated for less than 12 months, the composite outcome was reached in fewer patients in the combination group than in the monotherapy group (7.1% vs 47.1 %, p = 0.021). CONCLUSION: In CD patients in maintenance treatment with infliximab, combination treatment does not seem to have benefits over infliximab monotherapy beyond 12 months of treatment duration.

Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.

How many biomarker measurements are needed to predict prognosis in Crohn's disease patients under infliximab?-A prospective study.

BACKGROUND: Timely stratification of Crohn's disease (CD) is essential for patients' management. The use of noninvasive accurate biomarkers is key to monitor treatment and to pursue mucosal healing, the ultimate treatment endpoint in CD. OBJECTIVE: We aimed to evaluate the performance of readily available biomarkers and develop risk matrices to predict CD progression. METHODS: Data from 289 CD patients receiving infliximab (IFX) maintenance therapy for 2 years was collected; those patients were included in DIRECT, a prospective multicenter observational study. Disease progression was evaluated using two composite outcomes incorporating clinical and drug-related factors, the first including IFX dose and/or frequency adjustments. Univariate and multivariable logistic regressions were used to calculate the odds ratios (OR) and to develop risk matrices. RESULTS: The isolated presence of anemia at least once during follow-up was a significant predictor of disease progression (OR 2.436 and 3.396 [p ≤ 0.001] for composite outcomes 1 and 2, respectively) regardless of confounding factors. Isolated highly elevated C-reactive protein (CRP; >10.0 mg/L) and fecal calprotectin (FC; >500.0 µg/g) in at least one visit were also significant predictors, while milder elevations (3.1-10.0 mg/L and 250.1-500.0 µg/g) were only relevant when detected in at least two visits (consecutive or not). The combination of biomarkers in risk matrices had good ability to predict progression; patients simultaneously presenting anemia, highly elevated CRP and FC at least once had 42%-63% probability of achieving the composite outcomes. CONCLUSION: The combined evaluation of hemoglobin, CRP, and FC in at least one time point and their incorporation into risk matrices seems to be the optimal strategy for CD management, as data from additional visits did not meaningfully influence the predictions and may delay decision-making.

The Influence of Smartphones on Adolescent Sleep: A Systematic Literature Review.

(1) Background: Sleep is considered to be a complex condition for human beings, with the aim of ensuring physical and psychological recovery. Technology, including the cell phone, is a tool for teenagers that ensures they are always available to interact, even at night. This study aims to understand the influence of the use of smartphones on adolescent sleep quality. (2) Methods: The guidelines proposed by the Joanna Briggs Institute were followed. The search was conducted in October 2022 through the EBSCOhost platform, with access to the CINAHL Complete and Medline databases and through the b-On database. (3) Results: The use of electronic equipment plays an important role in adolescents' lives. There is a negative relationship between the use of electronic equipment, such as smartphones, and sleep, for reducing both the quality and quantity of sleep. There is also a relationship between nighttime smartphone use, insufficient sleep, and mental health problems. (4) Conclusions: The use of new technologies at night causes a change in the behavior of adolescents with repercussions in terms of the quality of sleep and sleep duration and consequent well-being and performance during the day.

Gait pattern analysis in the home environment as a key factor for the reliable assessment of shunt responsiveness in patients with idiopathic normal pressure hydrocephalus.

Background: The identification of patients with gait disturbance associated with idiopathic normal pressure hydrocephalus (iNPH) is challenging. This is due to the multifactorial causes of gait disturbance in elderly people and the single moment examination of laboratory tests. Objective: We aimed to assess whether the use of gait sensors in a patient's home environment could help establish a reliable diagnostic tool to identify patients with iNPH by differentiating them from elderly healthy controls (EHC). Methods: Five wearable inertial measurement units were used in 11 patients with iNPH and 20 matched EHCs. Data were collected in the home environment for 72 h. Fifteen spatio-temporal gait parameters were analyzed. Patients were examined preoperatively and postoperatively. We performed an iNPH sub-group analysis to assess differences between responders vs. non-responders. We aimed to identify parameters that are able to predict a reliable response to VP-shunt placement. Results: Nine gait parameters significantly differ between EHC and patients with iNPH preoperatively. Postoperatively, patients with iNPH showed an improvement in the swing phase (p = 0.042), and compared to the EHC group, there was no significant difference regarding the cadence and traveled arm distance. Patients with a good VP-shunt response (NPH recovery rate of ≥5) significantly differ from the non-responders regarding cycle time, cycle time deviation, number of steps, gait velocity, straight length, stance phase, and stance to swing ratio. A receiver operating characteristic analysis showed good sensitivity for a preoperative stride length of ≥0.44 m and gait velocity of ≥0.39 m/s. Conclusion: There was a significant difference in 60% of the analyzed gait parameters between EHC and patients with iNPH, with a clear improvement toward the normalization of the cadence and traveled arm distance postoperatively, and a clear improvement of the swing phase. Patients with iNPH with a good response to VP-shunt significantly differ from the non-responders with an ameliorated gait pattern.

Dipeptidyl peptidase 4 (DPP4) in fecal samples: validation of the extraction methodology and stability in short-term storage conditions.

OBJECTIVES: This study assesses the clinical relevance of dipeptidyl peptidase 4 (DPP4) membrane exopeptidase as a biomarker of inflammatory bowel disease (IBD). A spike-and-recovery approach of DPP4 in fecal samples was used to compare two different methods for protein extraction, followed by a stability assessment. METHODS: Fecal samples of healthy volunteers spiked with known concentrations of recombinant DPP4 were processed using a standard manual extraction protocol and the CALEX® protocol. The two methods were compared by quantification of fecal DPP4 by ELISA, followed by Bland-Altman analysis. For the stability assays DPP4 was extracted from fecal samples and stored under different conditions of temperature and time after collection. RESULTS: In general, the levels of spiked DPP4 in stool samples were lower with the manual protocol than in those obtained with the CALEX® method; this trend was corroborated by Bland-Altman analysis. Nonetheless, variability was within the acceptable limits for both protocols. In the stability assessment, no statistically significant differences were found between the results obtained under the different storage conditions. CONCLUSIONS: Both manual and CALEX® protocols provided equal extraction ability of DPP4 from stool samples. In addition, DPP4 provided flexibility in terms of sample storage enabling the accurate assessment of samples delivered up to a week before analysis.

Different biological effects of exposure to far-UVC (222 nm) and near-UVC (254 nm) irradiation.

Ultraviolet C (UVC) light has long been used as a sterilizing agent, primarily through devices that emit at 254 nm. Depending on the dose and duration of exposure, UV 254 nm can cause erythema and photokeratitis and potentially cause skin cancer since it directly modifies nitrogenated nucleic acid bases. Filtered KrCl excimer lamps (emitting mainly at 222 nm) have emerged as safer germicidal tools and have even been proposed as devices to sterilize surgical wounds. All the studies that showed the safety of 222 nm analyzed cell number and viability, erythema generation, epidermal thickening, the formation of genetic lesions such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs) and cancer-inducing potential. Although nucleic acids can absorb and be modified by both UV 254 nm and UV 222 nm equally, compared to UV 254 nm, UV 222 nm is more intensely absorbed by proteins (especially aromatic side chains), causing photooxidation and cross-linking. Here, in addition to analyzing DNA lesion formation, for the first time, we evaluated changes in the proteome and cellular pathways, reactive oxygen species formation, and metalloproteinase (MMP) levels and activity in full-thickness in vitro reconstructed human skin (RHS) exposed to UV 222 nm. We also performed the longest (40 days) in vivo study of UV 222 nm exposure in the HRS/J mouse model at the occupational threshold limit value (TLV) for indirect exposure (25 mJ/cm2) and evaluated overall skin morphology, cellular pathological alterations, CPD and 6-4PP formation and MMP-9 activity. Our study showed that processes related to reactive oxygen species and inflammatory responses were more altered by UV 254 nm than by UV 222 nm. Our chronic in vivo exposure assay using the TLV confirmed that UV 222 nm causes minor damage to the skin. However, alterations in pathways related to skin regeneration raise concerns about direct exposure to UV 222 nm.

Effect of antisiphon devices on ventriculoperitoneal shunt drainage dynamics in growing children.

OBJECTIVE: Infants and small children face changing boundary conditions when treated with a ventriculoperitoneal shunt (VPS) for hydrocephalus. There are no systematic data describing shunt drainage behavior and changes over time in a growing child. Using a child-adapted patient simulator, the authors investigated the drainage behaviors of fixed differential pressure (DP) valves and adjustable valves with devices for preventing overdrainage in children of different ages. METHODS: Three miniNAV DP valves with a 10-cm H2O medium-pressure setting (MN10) and three adjustable proGAV2.0 valves with a 25-cm H2O gravitational unit (GU) at low 5-cm H2O opening pressure (PG5) and medium 10-cm H2O opening pressure (PG10) settings were each investigated with a hardware-in-the-loop test bed. This test bed consisted of a posture motion mechanism and two pressure compartments that mimicked intracranial and abdominal pressures and was used to test the VPS under realistic in vitro conditions. Body orientation and length were physically set according to the child's age. The software simulated the physiological situations of children aged 1, 5, and 10 years. All valves were tested according to these specifications, with 5 runs for 1 hour each in the horizontal, vertical, and horizontal positions. Intracranial pressure (ICP) and VPS flow were measured, and the respective cerebrospinal fluid volume changes and ICP set value were computed. RESULTS: The drainage parameters increased with age in all valves in the vertical position, with that of MN10 being pronounced in the 1-year-old simulation. The GU values in PG5 and PG10 substantially reduced drainage compared with MN10. PG10 prevented drainage in the 1-year-old and 5-year-old setups, but there was some drainage at physiological ICP in the 10-year-old setup. In contrast, MN10 produced the largest decreases in ICP across all ages and positions, and overdrainage resulted in insufficient ICP recovery in the subsequent horizontal position. ICP levels were mostly constant with PG10 at all ages. CONCLUSIONS: This study shows that unprotected DP valves may lead to overdrainage in infants, whereas low-pressure GU valves can prevent overdrainage through 5 years and medium-pressure GU valves admit physiological ICP through at least 10 years. Therefore, devices for preventing overdrainage should be included in the first implanted shunt, and opening pressure should be adjusted as the child grows.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

UV 254 nm is more efficient than UV 222 nm in inactivating SARS-CoV-2 present in human saliva.

Ultraviolet (UV) light can inactivate SARS-CoV-2. However, the practicality of UV light is limited by the carcinogenic potential of mercury vapor-based UV lamps. Recent advances in the development of krypton chlorine (KrCl) excimer lamps hold promise, as these emit a shorter peak wavelength (222 nm), which is highly absorbed by the skin's stratum corneum and can filter out higher wavelengths. In this sense, UV 222 nm irradiation for the inactivation of virus particles in the air and surfaces is a potentially safer option as a germicidal technology. However, these same physical properties make it harder to reach microbes present in complex solutions, such as saliva, a critical source of SARS-CoV-2 transmission. We provide the first evaluation for using a commercial filtered KrCl excimer light source to inactivate SARS-CoV-2 in saliva spread on a surface. A conventional germicidal lamp (UV 254 nm) was also evaluated under the same condition. Using plaque-forming units (PFU) and Median Tissue Culture Infectious Dose (TCID50) per milliliter we found that 99.99% viral clearance (LD99.99) was obtained with 106.3 mJ/cm2 of UV 222 nm for virus in DMEM and 2417 mJ/cm2 for virus in saliva. Additionally, our results showed that the UV 254 nm had a greater capacity to inactivate the virus in both vehicles. Effective (after discounting light absorption) LD99.99 of UV 222 nm on the virus in saliva was ∼30 times higher than the value obtained with virus in saline solution (PBS), we speculated that saliva might be protecting the virus from surface irradiation in ways other than just by intensity attenuation of UV 222 nm. Due to differences between UV 222/254 nm capacities to interact and be absorbed by molecules in complex solutions, a higher dose of 222 nm will be necessary to reduce viral load in surfaces with contaminated saliva.

Leptin Signaling Suppression in Macrophages Improves Immunometabolic Outcomes in Obesity.

Obesity is a major concern for global health care systems. Systemic low-grade inflammation in obesity is a major risk factor for insulin resistance. Leptin is an adipokine secreted by the adipose tissue that functions by controlling food intake, leading to satiety. Leptin levels are increased in obesity. Here, we show that leptin enhances the effects of LPS in macrophages, intensifying the production of cytokines, glycolytic rates, and morphological and functional changes in the mitochondria through an mTORC2-dependent, mTORC1-independent mechanism. Leptin also boosts the effects of IL-4 in macrophages, leading to increased oxygen consumption, expression of macrophage markers associated with a tissue repair phenotype, and wound healing. In vivo, hyperleptinemia caused by diet-induced obesity increases the inflammatory response by macrophages. Deletion of leptin receptor and subsequently of leptin signaling in myeloid cells (ObR-/-) is sufficient to improve insulin resistance in obese mice and decrease systemic inflammation. Our results indicate that leptin acts as a systemic nutritional checkpoint to regulate macrophage fitness and contributes to obesity-induced inflammation and insulin resistance. Thus, specific interventions aimed at downstream modulators of leptin signaling may represent new therapeutic targets to treat obesity-induced systemic inflammation.

Spinous-Process-Splitting Versus Conventional Decompression for Lumbar Spinal Stenosis: Comparative Study with Respect to Short-Term Postoperative Pain and Analgesics Use.

OBJECTIVE: Several microsurgical techniques are available for the decompression of lumbar spinal stenosis (LSS). More recently, a spinous process-splitting laminectomy (SPSL) technique was introduced, with the premise of diminishing paraspinal muscle damage. This study aims to compare the neurologic and functional outcomes, as well as the differences in early postoperative pain and analgesic use during hospitalization after conventional decompression (CD) versus SPSL surgery for LSS. METHODS: Single-center retrospective analysis of all spinal decompression procedures (CD or SPSL) that were performed or supervised by one consulting spine surgeon, performed for LSS between 2015 and 2020. Preoperative neurologic symptoms, functional outcomes, as well as perioperative analgesic use and reported pain scales during hospitalization were analyzed. RESULTS: From a total of 106 patients, 58 were treated using CD and 48 using SPSL. In both groups, around one-third of the patients were taking opiates preoperatively (38% for CD, 31% for SPSL). Patients submitted to SPSL reported more pain on first postoperative day but significantly less pain in the further postoperative course (day 3 numeric rating scale [NRS] 2.4 vs. 3.4, P = 0.03 and on day 5 NRS 2.5 vs. 3.7, P = 0.009). Equal or less cumulative doses of analgesics were administered postoperatively (significantly less paracetamol on day 5 compared with CD; P = 0.013). Both groups showed a similarly favorable outcome in terms of improved mobility and there were no significant differences between complications and re-stenosis rates between both techniques. CONCLUSIONS: Patients treated with SPSL technique for LSS showed an equivalent favorable functional outcome compared to CD. However, SPSL patients showed significantly less subacute postoperative pain while using equal amounts or fewer analgesics postoperatively.

Subclinical Persistent Inflammation as Risk Factor for Crohn's Disease Progression: Findings From a Prospective Real-World Study of 2 Years.

BACKGROUND AND AIMS: Subclinical intestinal inflammation is common in Crohn's disease (CD). We aimed to explore its impact in the disease progression of infliximab-treated patients and the usefulness of fecal calprotectin (FC) and C-reactive protein (CRP) as surrogate minimally invasive biomarkers. METHODS: The registry-based, prospective, observational, multicenter DIRECT (study to investigate the correlation of fecal calprotectin with serum Drug levels and development of an antI-dRug antibodiEs among adult patients with inflammatory bowel disease reCeiving anti-TNF-alfa treatment or vedoluzimab treatment) study followed infliximab-treated CD patients for 2 years in a tertiary care setting. Persistent inflammation definition was based on FC (>150 µg/g, >250 µg/g, or >350 µg/g) or serum CRP (>3 µg/mL) concentrations over 2 consecutive or at least 3 visits. Patients were categorized according to a composite outcome reflecting disease progression that incorporated surgery; hospitalizations; new fistulae, abscess, or stricture; and treatment escalation. RESULTS: Of 322 DIRECT study patients, 180 asymptomatic, infliximab treated on maintenance regimen were included in the analysis. Patients developing the composite endpoint (n = 96) presented higher median levels of FC (205 [interquartile range, 98-515] µg/g; P = .045) but not of CRP (2.50 [interquartile range, 0.80-6.00] µg/mL; P = .895). Biomarker-defined persistent subclinical inflammation prevalence ranged from 24% to 81%. Considering FC >250 µg/g in 2 consecutive visits, prevalence was 50%, odds of achieving the endpoint were increased 3-fold (odds ratio, 2.996 [95% confidence interval, 1.557-5.776]), and time-to-outcome occurrence was significantly lower among subjects with persistent inflammation (median time: 11 months). Both clinical-related and treatment-related components were significantly associated with persistent inflammation. Definitions based on CRP >3 µg/mL, FC >150 µg/g, FC >350 µg/g, double biomarkers (FC >250 µg/g and/or CRP >3 µg/mL), or more visits did not improve predictive ability. CONCLUSIONS: Persistent inflammation, defined simply and readily by FC >250 µg/g over 2 consecutive visits, was associated with a significantly higher risk and shorter time to occurrence of a composite outcome reflecting disease progression in asymptomatic infliximab-treated CD patients.

High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules.

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays. A structure-activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity.