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Embryonic stem cells (ESCs) are remarkable for the high activity level of ubiquitin-proteasome system-the molecular machinery of protein degradation in the cell. Various forms of the proteasome complexes comprising different subunits and interacting regulators are responsible for the substrate selectivity and degradation. Immunoproteasomes are amongst these forms which play an important role in antigen presentation; however, a body of recent evidence suggests their functions in pluripotent stem cells. Previous studies have established three consecutive phases of pluripotency, featured by epiblast cells and their cultured counterparts: naïve, formative, and primed phase. In this work, we report that immunoproteasomes and their chaperone co-regulators are suppressed in the naïve state but are readily upregulated in the formative phase of the pluripotency continuum, featured by epiblast-like cells (EpiLCs). Our data lay ground for the further investigation of the biological functions of immunoproteasome in the regulation of proteostasis during early mammalian development.

In recent years, genome and transcriptome mining have dramatically expanded the rate of discovering diverse natural products from bacteria and fungi. In plants, this approach is often more limited due to the lack of available annotated genomes and transcriptomes combined with a less consistent clustering of biosynthetic genes. The recently identified burpitide class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products offer a valuable opportunity for bioinformatics-guided discovery in plants due to their short biosynthetic pathways and gene encoded substrates. Using a high-throughput approach to assemble and analyze 700 publicly available raw transcriptomic data sets, we uncover the potential distribution of split burpitide precursor peptides in Streptophyta. Metabolomic analysis of target plants confirms our bioinformatic predictions of new cyclopeptide alkaloids from both known and new sources., (Copyright © 2024, Kriger et al.)

Background: ACTN4 is an actin-binding protein involved in many cellular processes, including cancer development. High ACTN4 expression is often associated with a poor prognosis. However, it has been identified as a positive marker for platinum-based adjuvant chemotherapy for non-small cell lung cancer (NSCLC). The goal of our study was to investigate the involvement of ACTN4 in the NSCLC cells' response to the genotoxic drugs., Results: We generated H1299 cells with the ACTN4 gene knock-out (ACTN4 KO), using the CRISPR/Cas9 system. The resistance of the cells to the cisplatin and etoposide was analyzed with the MTT assay. We were also able to estimate the efficiency of DNA repair through the DNA comet assay and gamma-H2AX staining. Possible ACTN4 effects on the non-homologous end joining (NHEJ) and homologous recombination (HR) were investigated using pathway-specific reporter plasmids and through the immunostaining of the key proteins. We found that the H1299 cells with the ACTN4 gene knock-out did not show cisplatin-resistance, but did display a higher resistance to the topoisomerase II inhibitors etoposide and doxorubicin, suggesting that ACTN4 might be somehow involved in the repair of DNA strand breaks. Indeed, the H1299 ACTN4 KO cells repaired etoposide- and doxorubicin-induced DNA breaks more effectively than the control cells. Moreover, the ACTN4 gene knock-out enhanced NHEJ and suppressed HR efficiency. Supporting the data, the depletion of ACTN4 resulted in the faster assembly of the 53BP1 foci with a lower number of the phospho-BRCA1 foci after the etoposide treatment., Conclusions: Thus, we are the first to demonstrate that ACTN4 may influence the resistance of cancer cells to the topoisomerase II inhibitors, and affect the efficiency of the DNA double strand breaks repair. We hypothesize that ACTN4 interferes with the assembly of the NHEJ and HR complexes, and hence regulates balance between these DNA repair pathways., (© 2022. The Author(s).)

Delayed presentation of acute appendicitis is associated with increased complications. We hypothesized that the outcomes of appendectomy in delayed presentations of acute appendicitis (>72 hours of pain) were dependent on radiologic findings rather than late presentation. We reviewed records from 2009 to 2015 and analyzed delayed presentations of acute appendicitis. We divided patients into three groups based on specific CT findings: uncomplicated appendicitis (UA), phlegmon or abscess (PA), and other perforated appendicitis (PERF, signs of perforation without abscess or phlegmon). One hundred thirty-eight patients were included in this study (58 in the UA, 67 in the PA, and 13 in the PERF groups). Overall, 78 (57%) patients underwent early appendectomy (EA) and 60 (43%) underwent initial conservative management. The incidence of adverse events was lower in EA than that in initial conservative management (17% vs 42%, P = 0.005). EA in the UA group was associated with shorter hospitalization (3.2 vs 5.6 days, P < 0.001) and less adverse events (6% vs 29%, P < 0.05). Severe adverse events (two colectomies and one fecal fistula) were observed in the PA group. In conclusion, in these late presentations of appendicitis, complicated appendicitis was common. EA was safe in selected patients, however, and associated with decreased adverse events.

The p53 protein is a key tumor suppressor in mammals. In response to various forms of genotoxic stress p53 stimulates expression of genes whose products induce cell cycle arrest and/or apoptosis. An E3-ubiquitin ligase, Mdm2 (mouse-double-minute 2) and its human ortholog Hdm2, physically interact with the amino-terminus of p53 to mediate its ubiquitin-mediated degradation via the proteasome. Thus, pharmacological inhibition of the p53-Mdm2 interaction leads to overall stabilization of p53 and stimulation of its anti-tumorigenic activity. In this study we characterize the biological effects of a novel class of non-genotoxic isatin Schiff and Mannich base derivatives (ISMBDs) that stabilize p53 on the protein level. The likely mechanism behind their positive effect on p53 is mediated via the competitive interaction with Mdm2. Importantly, unlike Nutlin, these compounds selectively promoted p53-mediated cell death. These novel pharmacological activators of p53 can serve as valuable molecular tools for probing p53-positive tumors and set up the stage for development of new anti-cancer drugs.

Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.

The use of ultrasound to diagnose appendicitis in pediatric patients has been growing with the improvement of ultrasound technology and operator skills, but its utility in the increasingly obese pediatric population has not been thoroughly investigated. A retrospective review of all pediatric (≤18 years old) patients with appendicitis who were admitted at a single hospital from 2014 to 2016 was conducted. Patients were stratified into body mass index (BMI) percentile categories based on the centers for disease control guidelines. Comparisons were then made. There were 231 patients with an average BMI percentile of 72.6; 99 (42.9%) who had an ultrasound, of which 54 (54.5%) were positive for acute appendicitis, whereas 43 (43.4%) were nondiagnostic. In patients who had a nondiagnostic ultrasound, 37 had a CT demonstrating acute appendicitis. These were compared with 123 patients who had CT alone demonstrating acute appendicitis. The CT-only group was older (12 vs 9, P < 0.005), tended to be male (78 (63%) vs 15 (41%), P = 0.019), had fewer operations performed (81 (66%) vs 30 (81%), P = 0.048) but had no significant difference in BMI percentile (75.8 vs 71.7, P = 0.465). Ultrasound had a 100 per cent positive predictive value in obese and overweight children. Ultrasound is a reliable study in obese and overweight pediatric patients with acute appendicitis.

Objectives: To evaluate the use of single-dose flumazenil in the diagnosis of coma of unknown etiology, and of continuous flumazenil infusion in the treatment of benzodiazepine-induced coma., Design: Prospective study., Setting: Emergency room and general medicine ward of a teaching hospital., Patients: A total of 42 comatose adults in whom metabolic, neurologic, or traumatic causes of coma were excluded., Interventions: a) Intravenous bolus injections of 0.25 mg flumazenil were given at 1-min intervals, either until improvement by two coma grades or a total dose of 2.0 mg was reached. b) Loading doses as in (a) followed by a maintenance infusion administered as long as indicated by repeated coma grade evaluation., Measurements and Main Results: a) Of 34 patients, 28 received only the flumazenil loading dose responded promptly. Twenty-one of 25 available urine samples of the responding patients contained only benzodiazepine metabolites. Four urine samples contained benzodiazepines in combination with other drugs. Six patients did not respond to the flumazenil loading dose. The urine of three patients contained a combination of benzodiazepines and another coma-exerting drug; the remaining three were negative. A total of 24 patients, who initially responded to flumazenil loading, deteriorated to their previous coma state and were admitted to the general medical ward. Six (25%) patients developed complications related to hospitalization and their bedridden state. b) Eight other patients, who deteriorated after an initial loading dose, received a second iv bolus of flumazenil, followed by maintenance infusions over 5 to 24 hrs. Their hospital course was uneventful., Conclusions: These findings indicate that flumazenil is safe and effective in the diagnosis of benzodiazepine-induced coma. Furthermore, the use of continuous flumazenil maintenance infusion is of considerable therapeutic value in patients who exhibit deterioration after initial response to the single loading dose.