Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN). The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes

High tumor mutational burden (TMB) identifies a microsatellite stable pancreatic cancer subset with prolonged survival and strong anti-tumor immunity.

AIM Tumor mutational burden (TMB: somatic mutations per megabase, mut/Mb) predicts the efficacy of immunotherapy. Here, we link TMB levels with the activation of immune pathways and intratumoral immune responses in pancreatic adenocarcinoma (PDAC) to explore immunoarchitectural patterns associated with high TMB. METHODS We assessed TMB in 161 resected, microsatellite stable (MSS) PDACs, including 41 long-term survivors (LTS). Five microsatellite instable (MSI-high) cases were also assessed. Cases were classified into TMB-high (≥10 mut/Mb), TMB-intermediate (>5 < 10 mut/Mb), and TMB-low (≤5 mut/Mb) categories. Tumors additionally underwent mRNA in situ hybridization for immune pathway genes and were immunoprofiled by multiplex immunofluorescence followed by automated image analysis. RESULTS We detected 12 TMB-high, 28 TMB-intermediate, and 121 TMB-low cases. TMB-high tumors comprised ten LTSs (10/41; 24%) and two conventional PDACs (2/120; 1.7%). They exhibited the highest T cell density with significantly increased CD3+CD4+T helper and CD208+dendritic cell (DC) counts, compared to all other cases. CD3+CD8+cytotoxic T cells were significantly closer to tumor cells and T helper cells closer to DCs in TMB-high PDACs. Immune pathways involved in T cell activation, immune cell adhesion/migration, antigen presentation, and cytokine signaling were upregulated in most TMB-high and many TMB-intermediate tumors. ARID1A and ERBB4 alterations were more frequent in TMB-high PDACs. All MSI-high PDACs were TMB-high. CONCLUSIONS TMB-high cases frequently belong to specific PDAC subsets with prolonged survival such as LTSs and MSI-high PDACs. They display strong anti-tumor immune responses fueled by a T helper cell/DC-mediated priming of the cytotoxic T cells. Moreover, they frequently harbor further actionable alterations

Organic synthesis in a modular robotic system driven by a chemical programming language

The synthesis of complex organic compounds is largely a manual process that is often incompletely documented. To address these shortcomings, we developed an abstraction that maps commonly reported methodological instructions into discrete steps amenable to automation. These unit operations were implemented in a modular robotic platform using a chemical programming language which formalizes and controls the assembly of the molecules. We validated the concept by directing the automated system to synthesize three pharmaceutical compounds, Nytol, rufinamide, and sildenafil, without any human intervention. Yields and purities of products and intermediates were comparable to or better than those achieved manually. The syntheses are captured as digital code that can be published, versioned, and transferred flexibly between platforms with no modification, thereby greatly enhancing reproducibility and reliable access to complex molecules

Assessing the risk of central post-stroke pain of thalamic origin by lesion mapping

Central post-stroke pain of thalamic origin is an extremely distressing and often refractory disorder. There are no well-established predictors for pain development after thalamic stroke, and the role of different thalamic nuclei is unclear. Here, we used structural magnetic resonance imaging to identify the thalamic nuclei, specifically implicated in the generation of central post-stroke pain of thalamic origin. Lesions of 10 patients with central post-stroke pain of thalamic origin and 10 control patients with thalamic strokes without pain were identified as volumes of interest on magnetic resonance imaging data. Non-linear deformations were estimated to match each image with a high-resolution template and were applied to each volume of interest. By using a digital atlas of the thalamus, we elucidated the involvement of different nuclei with respect to each lesion. Patient and control volumes of interest were summed separately to identify unique areas of involvement. Voxelwise odds ratio maps were calculated to localize the anatomical site where lesions put patients at risk of developing central post-stroke pain of thalamic origin. In the patients with pain, mainly lateral and posterior thalamic nuclei were affected, whereas a more anterior-medial lesion pattern was evident in the controls. The lesions of 9 of 10 pain patients overlapped at the border of the ventral posterior nucleus and the pulvinar, coinciding with the ventrocaudalis portae nucleus. The lesions of this area showed an odds ratio of 81 in favour of developing thalamic pain. The high odds ratio at the ventral posterior nucleus-pulvinar border zone indicates that this area is crucial in the pathogenesis of thalamic pain and demonstrates the feasibility of identifying patients at risk of developing central post-stroke pain of thalamic origin early after thalamic insults. This provides a basis for pre-emptive treatment studie

Artificial intelligence large language model ChatGPT: is it a trustworthy and reliable source of information for sarcoma patients?

IntroductionSince its introduction in November 2022, the artificial intelligence large language model ChatGPT has taken the world by storm. Among other applications it can be used by patients as a source of information on diseases and their treatments. However, little is known about the quality of the sarcoma-related information ChatGPT provides. We therefore aimed at analyzing how sarcoma experts evaluate the quality of ChatGPT’s responses on sarcoma-related inquiries and assess the bot’s answers in specific evaluation metrics.MethodsThe ChatGPT responses to a sample of 25 sarcoma-related questions (5 definitions, 9 general questions, and 11 treatment-related inquiries) were evaluated by 3 independent sarcoma experts. Each response was compared with authoritative resources and international guidelines and graded on 5 different metrics using a 5-point Likert scale: completeness, misleadingness, accuracy, being up-to-date, and appropriateness. This resulted in maximum 25 and minimum 5 points per answer, with higher scores indicating a higher response quality. Scores ≥21 points were rated as very good, between 16 and 20 as good, while scores ≤15 points were classified as poor (11–15) and very poor (≤10).ResultsThe median score that ChatGPT’s answers achieved was 18.3 points (IQR, i.e., Inter-Quartile Range, 12.3–20.3 points). Six answers were classified as very good, 9 as good, while 5 answers each were rated as poor and very poor. The best scores were documented in the evaluation of how appropriate the response was for patients (median, 3.7 points; IQR, 2.5–4.2 points), which were significantly higher compared to the accuracy scores (median, 3.3 points; IQR, 2.0–4.2 points; p = 0.035). ChatGPT fared considerably worse with treatment-related questions, with only 45% of its responses classified as good or very good, compared to general questions (78% of responses good/very good) and definitions (60% of responses good/very good).DiscussionThe answers ChatGPT provided on a rare disease, such as sarcoma, were found to be of very inconsistent quality, with some answers being classified as very good and others as very poor. Sarcoma physicians should be aware of the risks of misinformation that ChatGPT poses and advise their patients accordingly

Phosphorylated Histone 3 at Serine 10 Identifies Activated Spinal Neurons and Contributes to the Development of Tissue Injury-Associated Pain

Transcriptional changes in superficial spinal dorsal horn neurons (SSDHN) are essential in the development and maintenance of prolonged pain. Epigenetic mechanisms including post-translational mo difications in histones are pivotal in regulating transcription. Here, we report th at phosphorylation of serine 10 (S10) in histone 3 (H3) specifically occurs in a group of rat SSDHN following the activation of nociceptive primary sensory neurons by burn injury, capsaicin application or sustained electrical activation of nociceptive primary sensory nerve fibres. In contrast, brief thermal or mechanical nociceptive stimuli, which fail to induce tissue injury or inflammation, do not produce the same effect. Blocking N-methyl-D-aspartate receptors or activation of extracellular signa l-regulated kinases 1 and 2, or blocking or deleting the mitogen- and stress-activated kinases 1 and 2 (MSK1/2), which phosphorylate S10 in H3, inhibit up-regulation in phosphorylated S10 in H3 ( p - S10H3) as well as fos transcription, a down-stream effect of p -S10H3. Deleting MSK1/2 also inhibits the development of carrageenan-induced inflammatory heat hyperalgesia in mice. We propose that p -S10H3 is a novel marker for nociceptive processing in SSDHN with high relevance to transcriptional changes and the development of prolonged pain

Excellent Response to OnabotulinumtoxinA : Different Definitions, Different Predictors

The identification of patients who can benefit the most from the available preventive treatments is important in chronic migraine. We explored the rate of excellent responders to onabotulinumtoxinA in a multicenter European study and explored the predictors of such response, according to different definitions. A pooled analysis on chronic migraineurs treated with onabotulinumtoxinA and followed-up for, at least, 9 months was performed. Excellent responders were defined either as patients with a ≥75% decrease in monthly headache days (percent-based excellent responders) or as patients with <4 monthly headache days (frequency-based excellent responders). The characteristics of excellent responders at the baseline were compared with the ones of patients with a <30% decrease in monthly headache days. Percent-based excellent responders represented about 10% of the sample, whilst frequency-based excellent responders were about 5% of the sample. Compared with non-responders, percent-based excellent responders had a higher prevalence of medication overuse and a higher excellent response rate even after the 1st and the 2nd injection. Females were less like to be frequency-based excellent responders. Chronic migraine sufferers without medication overuse and of female sex may find fewer benefits with onabotulinumtoxinA. Additionally, the excellent response status is identifiable after the first cycle