Although vagus nerve stimulation (VNS) is widely used, therapeutic mechanisms and optimal stimulation parameters remain elusive. In the present study, we investigated the effect of VNS on hippocampal ...field activity and compared the efficiency of different VNS paradigms. Hippocampal electroencephalography (EEG) and perforant path dentate field-evoked potentials were acquired before and during VNS in freely moving rats, using 2 VNS duty cycles: a rapid cycle (7 s on, 18 s off) and standard cycle (30 s on, 300 s off) and various output currents. VNS modulated the evoked potentials, reduced total power of the hippocampal EEG, and slowed the theta rhythm. In the hippocampal EEG, theta (4–8 Hz) and high gamma (75–150 Hz) activity displayed strong phase amplitude coupling that was reduced by VNS. Rapid-cycle VNS had a greater effect than standard-cycle VNS on all outcome measures. Using rapid cycle VNS, a maximal effect on EEG parameters was found at 300 μA, beyond which effects saturated. The findings suggest that rapid-cycle VNS produces a more robust outcome than standard cycle VNS and support already existing preclinical evidence that relatively low output currents are sufficient to produce changes in brain physiology and thus likely also therapeutic efficacy.
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Background: Biomarker analysis predicting outcome with neoadjuvant atezolizumab in muscle invasive bladder cancer (MIBC) was performed. This includes previously unreported findings ...with FOXP3 and MHC1. Methods: Sequential tissue obtained from 95 patients in the ABACUSstudy (NCT02662309) which investigated two cycles of atezolizumab prior to cystectomy in MIBC patients. Multiplex immunohistochemistry staining, Foundation One analysis and RNA sequencing was performed on paired pre- and post- treatment samples. CD8+ T cells, fibroblast activation protein (FAP), granzyme – B (GZMB), FOXP3, MHC1, PD-L1 expressions were analyzed and correlated with outcome. Results: After a median follow up of 13.1 months, 31% had a pathological complete response (pCR) and 18% have relapsed. 40% were PD-L1 positive at baseline and 73%, 19% and 8%had inflamed, excluded and desert phenotype respectively. CD8/GZMB and CD8/MHC1 expression prior to therapy was significantly associated with response in inflamed tumors. CD8, PDL1, GZMB and MHC1 expression all increased with treatment in this group of patients. Immune desert phenotype at baseline, showed low and static expression of immune biomarker. TGFb correlated with relapse in excluded phenotypes. Treatment was associated with increase in cell cycle genes and FAP, both of which were associated with relapse. FOXP3 expression correlated with CD8 and increased with therapy in responding tumors. Combining FAP (high) to CD8 (low) and MHC1 (low) increased the positive predictive value for relapse. TMB did not correlate with outcome or increase with therapy. FGF DNA alterations were associated with response. Conclusions: MIBCs have high T effector immune expression, which may account for high pCR rates. Biomarkers at baseline and after treatment can predict outcome. Assessment of multiple biomarker within algorithms improves accuracy in predicting outcome. FOXP3 expression correlates with activated T cell expression, potentially accounting for the counterintuitive findings. Clinical trial information: NCT02662309.
Fibroblast activation protein-α (FAP) is a potential prognostic biomarker for progression in patients with high-risk non–muscle-invasive bladder cancer treated with bacillus Calmette-Guérin.
The ...tumor microenvironment (TME) in non–muscle-invasive bladder cancer (NMIBC) plays an important role in the anticancer response. We aimed to identify the prognostic biomarkers in the TME of patients with NMIBC for progression to ≥T2.
From our institutional database, 40 patients with T1 high-risk NMIBC who progressed were pair matched for Club Urologico Español de Tratamiento Oncologico (CUETO) progression variables with 80 patients who never progressed despite longer follow-up. Progression was defined as ≥T2 or extravesical disease. Patients were treated at least with bacillus Calmette-Guérin (BCG) induction (five or more of six doses). Immunohistochemical (IHC) markers for the TME were used on tissue at first T1 diagnosis: CD8-PanCK, GZMB-CD8-FOXP3, CD163, PD-L1 SP142/SP263, fibroblast activation protein-α (FAP), and CK5-GATA3. Full tissue slides were annotated digitally. Relative marker area (IHC-positive area/total area) or density (IHC-positive cells per area; n/mm2) was calculated, differentiating between regions of interest (ROIs; T1, Ta, and carcinoma in situ) and between compartments (stromal, epithelial, and combined). Differences in IHC variables were assessed using the t test, for continuous variables using analysis of variance and comparisons of more than two groups using Tukey’s test. Conditional logistic regression for progression at 5-yr follow-up was performed with clusters based on pair matching.
Only FAP expression (increase per 50%) in T1 (odds ratio OR: 1.33; 95% confidence interval CI: 1.04–1.70) and all ROIs combined (OR: 1.62; 95% CI: 1.14–2.29) correlated significantly with progression. None of the other clinicopathological/IHC variables correlated with progression.
FAP is a potential prognostic biomarker for progression in high-risk NMIBC. FAP is a marker for cancer-associated fibroblasts and is linked to immunosuppression and neoangiogenesis, which makes future investigation clinically relevant.
We found that progression of high-risk non–muscle-invasive bladder cancer to muscle-invasive disease is less in patients with lower fibroblast activation protein-α (FAP) expression, which is a marker for cancer-associated fibroblasts.
Up to 50% of uveal melanomas (UM) metastasise to the liver within 10 years of diagnosis, and these almost always prove rapidly fatal. As histopathological growth patterns (HGPs) of liver metastases ...of the replacement and desmoplastic type, particularly from colon and breast carcinoma, may import valuable biological and prognostic information, we have studied HGP in a series of 41 UM liver metastases originating from 41 patients from the period 2006–2017. Twenty patients underwent enucleation while 21 had radiation therapy. Analysis of UM by array comparative genomic hybridisation revealed: 25 (64%) patients with high risk (monosomy3/8q gain); 13 (33%) intermediate risk (M3/8normal or disomy3/8q gain); and 1 low risk (disomy3/8normal). The principal HGP was replacement in 30 (73%) cases and desmoplastic in 11 (27%) cases. Cases with replacement demonstrated striking vascular co‐option/angiotropism. With the development of liver metastasis, only the replacement pattern, largest primary tumour diameter, and R2 (incomplete resection) status predicted diminished overall survival (OS; p < 0.041, p < 0.017, p < 0.047, respectively). On multivariate analysis, only HGP (hazard ratio; HR = 6.51, p = 0.008) and resection status remained significant. The genomic high‐risk variable had no prognostic value at this stage of liver metastasis. Chi‐square test showed no association of HGP with monosomy 3 or 8q gain. Eighteen of 41 (44%) patients are alive with disease and 23 (56%) patients died with follow‐up ranging from 12 to 318 months (mean: 70 months, median: 47 months). In conclusion, we report for the first time the frequency of the replacement and desmoplastic HGPs in liver UM metastases resected from living patients, and their potential important prognostic value for UM patients, as in other solid cancers. These results may potentially be utilised to develop radiological correlates and therapeutic targets for following and treating patients with UM metastases.
Globally, over 50 million people are affected by epilepsy, which is characterized by the occurrence of spontaneous recurrent seizures. Almost one-third of the patients show resistance to current ...anti-epileptic drugs, making the exploration of new molecular targets necessary. An interesting target may be Homer1, due to its diverse roles in epileptogenesis and synaptic plasticity. Indeed, Homer1 regulates group I metabotropic glutamate (mGlu) receptors (i.e. mGlu
and mGlu
) scaffolding and signaling in neurons. In the present work, using the systemic kainic acid (KA)-induced status epilepticus (SE) model in adult rats, we investigated the mRNA and protein expression patterns of the mGlu
receptor, Homer1a and Homer1b/c at 10, 80 and 120 days post-SE (i.e. T10, T80 and T120). Epileptogenesis was validated by electrophysiological recordings of seizures via electroencephalography (EEG) monitoring and through upregulation of glial fibrillary acidic protein. At the protein level, the mGlu
receptor was downregulated in the late latent phase (T10) and the early- and late exponential growth phase (T80 and T120, respectively), which was best observed in the hippocampal CA1 region. At mRNA level, significant downregulation of the mGlu
receptor was only detected in the late exponential growth phase. Homer1a expression did not change at any investigated time point. Interestingly, Homer1b/c was only downregulated in the late latent phase, a period where spontaneous seizures are extremely rare. Thus, this phase-specific downregulation may be indicative of an endogenous neuroprotective mechanism. In conclusion, these results suggest that Homer1b/c may be an interesting molecular target to prevent epileptogenesis and/or control seizures.
OBJECTIVEThis study was designed to evaluate the dialysability of gadoteric acid in patients with end-stage renal disease (ESRD) requiring hemodialysis. Gadoteric acid is used for magnetic resonance ...imaging. It is cleared from the blood exclusively by glomerular filtration. In an attempt to decrease the occurrence of nephrogenic systemic fibrosis, hemodialysis has been advocated immediately after injection of gadolinium-based contrast agents in patients with ESRD.
METHODSTen patients with ESRD underwent 3 consecutive 4-hour hemodialysis sessions after a single intravenous injection (0.1 mmol/kg) of gadoteric acid. Blood samples were drawn from the vascular access just before and immediately after the end of each 4-hour session. Additional sampling was performed during session 1.
RESULTSGadoteric acid was efficiently eliminated, with mean (95% confidence interval) clearance values (mL/min) of 224.6 (216.0–233.9) at 0.5 hours and 225.9 (215.6–237.2) at 1.5 hours during session 1. The gadolinium concentration decrease after a 4-hour hemodialysis (vs the corresponding predialysis values) was 97% after session 1. The decrease in gadolinium concentration after session 3 was 99.7% compared with the predialysis concentration of session 1. These percentages are similar to those reported after hemodialysis for other gadolinium-based contrast agents. No adverse effects related to gadoteric acid were reported.
CONCLUSIONAlthough no unconfounded case of nephrogenic systemic fibrosis has been reported so far after injection of gadoteric acid, hemodialysis can be efficiently used to remove this molecule in patients with ESRD already undergoing long-term hemodialysis.
In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with ...severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.
Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is ...responsible for the biosynthesis of the oximidine anticancer agents, oxime‐substituted benzolactone enamides that inhibit vacuolar H+‐ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O‐methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans‐AT PKSs and identify potential strategies for the production of novel oximidine analogues.
The anticancer agent oximidine I and three novel variants were discovered as products of a cryptic trans‐AT PKS/NRPS in Pseudomonas baetica. By manipulating the biosynthetic pathway, a key intermediate was identified that retains potent anticancer properties. A combination of bioinformatics analysis and genetic and biochemical experiments illuminated the oximidine biosynthetic pathway, including a novel mechanism for O‐methyloxime formation.
Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is ...responsible for the biosynthesis of the oximidine anticancer agents, oxime‐substituted benzolactone enamides that inhibit vacuolar H+‐ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O‐methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans‐AT PKSs and identify potential strategies for the production of novel oximidine analogues.
The anticancer agent oximidine I and three novel variants were discovered as products of a cryptic trans‐AT PKS/NRPS in Pseudomonas baetica. By manipulating the biosynthetic pathway, a key intermediate was identified that retains potent anticancer properties. A combination of bioinformatics analysis and genetic and biochemical experiments illuminated the oximidine biosynthetic pathway, including a novel mechanism for O‐methyloxime formation.
Abstract
In synthetic biology, small molecules are commonly used for altering gene expression through the (in)activation of gene switches. Current switches, however, have a narrow species range, are ...non-titratable, or use non-inert inducers which evoke important side effects. Universal gene switches (XRs) that have dynamic ranges without interference with the host cell physiology will overcome these limitations. Ideal starting points for developing such XRs are the steroid receptors (SRs). The SRs are frequently applied as regulator domain or gene switch in organisms in which they are not naturally expressed, like microbes and plants. Several SRs, such as the estrogen receptor alpha (ERα), have been redesigned to respond to synthetic effectors but these variants require inducer amounts that are detrimental to the host or affect endogenous SRs.
By combining in silico modelling with directed evolution in the yeast Saccharomyces cerevisiae, we developed a platform and strategy for evolving SRs towards a chemical of choice. Validation of this method was achieved by first evolving the ERα to employ tamoxifen as an agonist, yielding the preTERRA variant. Subsequently, activation by estradiol was removed, rendering a tamoxifen receptor (TERRA). We show that TERRA gets activated by subtoxic tamoxifen amounts while remaining insensitive for physiological estradiol levels. Further evaluation demonstrated TERRA to be transferable to mammalian cells. Ongoing research aims at converting the TERRA receptor into a regulatory domain for controlling nuclear translocation of Cre recombinases and Cas9 proteins in both cell lines and mouse models.
The same platform was employed for adapting ERα towards the plasticizer Bisphenol A. This yielded an ERα variant that is no longer inducible by estradiol but is responsive to lower concentrations of Bisphenol in yeast. The transferal to cell lines will provide us with an additional regulatory domain. The ability to engineer any SR towards a synthetic ligand of choice not only expands the synthetic biology toolbox but can find applications in landscaping SR-related drug resistance and ligand-inducibility.
Presentation: Saturday, June 11, 2022 1:48 p.m. - 1:53 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.
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