Label-free detection of prostaglandin transporter (SLCO2A1) function and inhibition: insights by wound healing and TRACT assays.

The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE2)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low extracellular PGE2 levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE2. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE2. PGE2 potency could be recovered upon inhibition of PGT-mediated PGE2 uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE2 and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies.

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

Background: In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. Methods: In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. Results: GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Conclusions: Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

Reduced ossification caused by 3D simulated microgravity exposure is short-term in larval zebrafish.

Understanding how skeletal tissues respond to microgravity is ever more important with the increased interest in human space travel. Here, we exposed larval Danio rerio at 3.5 dpf to simulated microgravity (SMG) using a 3D mode of rotation in a ground-based experiment and then studied different cellular, molecular, and morphological bone responses both immediately after exposure and one week later. Our results indicate an overall decrease in ossification in several developing skeletal elements immediately after SMG exposure with the exception of the otoliths, however ossification returns to normal levels seven days after exposure. Coincident with the reduction in overall ossification tnfsf11 (RANKL) expression is highly elevated after 24 h of SMG exposure and also returns to normal levels seven days after exposure. We also show that genes associated with osteoblasts are unaffected immediately after SMG exposure. Thus, the observed reduction in ossification is primarily the result of a high level of bone resorption. This study sheds insight into the nuances of how osteoblasts and osteoclasts in the skeleton of a vertebrate organism respond to an external environmental disturbance, in this case simulated microgravity.

The Need to Appear Healthy: Concealment of Chronic Illness, Privacy, and Self-Sufficiency Among Chronically Ill Older Nigerians.

Background and Objectives: Prior research has highlighted the beneficial impact of social networks and social support on older adults' physical and psychosocial well-being. However, the impact of the relationship between chronic illness and social networks on the psychosocial well-being of older Nigerians remains understudied. This study explored how older Nigerians with chronic illnesses navigate the physical, mental, and emotional changes due to their chronic disease diagnosis within their social contexts. Research Design and Methods: The current qualitative study used semistructured in-depth interviews with 19 purposively sampled older adults, aged 50 years and over, chronically ill, and receiving clinical care to examine the role of social networks in how chronically ill older Nigerians cope with their diagnosis. Results: Three main themes reflecting participants' experiences emerged from the findings: (1) closely knit circles, (2) privacy and self-sufficiency, and (3) body image. Results show that chronically ill older Nigerians prefer to keep the knowledge of their conditions strictly within their close family circles. It was considered horrific to inform friends, community members, and religious groups about one's chronic illness. Findings further reveal that the need to appear healthy to one's social network stems from the fear of being discriminated against and attempts to maintain some level of normalcy when interacting with others. Additionally, feelings of inferiority and shame limited their participation in social activities and social network maintenance. Discussion and Implications: We discuss the implications of the results for the mental well-being and quality of life of chronically ill older Nigerians and make recommendations for policies and resources that can improve the well-being of chronically ill Nigerians.

Teaching Zebrafish Development in a STEM-Based Science Camp for Middle School Students.

Zebrafish have been used as an education tool for students of all ages and can be used in many learning environments to teach different fields of science. In this study, we focus on the biology of zebrafish. We describe an educational program within a weeklong science camp for students between 12 and 14 years old. The methodology described is based on running annual science camps over an 11-year period. In these camps, students learnt about the developmental stages of zebrafish, as well as general zebrafish biology, husbandry, ecology, behavior, and reproduction. This article describes how to provide students and educators with an educational program to explore, discover, and contribute to the ever-evolving landscape of biological understanding through active and visual learning. We describe the methodology, the evaluation, revisions to our program over time, and future directions for expansion.

A Bibliometric Analysis of Study of Associations of Certain Genotypes with the Cardiovascular Form of Diabetic Neuropathy.

This bibliometric analysis explores the landscape of research on the associations between specific genotypes and the cardiovascular form of diabetic neuropathy. Diabetes mellitus (DM) is a major contributor to premature mortality, primarily due to increased susceptibility to cardiovascular diseases. The global prevalence of DM is rising, with projections indicating further increases. Diabetic neuropathy, a complication of DM, includes the cardiovascular subtype, posing challenges in diagnosis and management. Understanding the genetic basis of cardiovascular diabetic neuropathy is crucial for targeted therapeutic interventions. The study utilizes bibliometric analysis to synthesize existing literature, identify trends, and guide future research. The Scopus database was searched, applying inclusion criteria for English articles related to genotypes and cardiovascular diabetic neuropathy. The analysis reveals a dynamic field with a notable impact, collaborative efforts, and multidimensional aspects. Publication trends over 1997-2023 demonstrate fluctuating research intensity. Top journals, authors, and affiliations are highlighted, emphasizing global contributions. Keyword analysis reveals thematic trends, and citation analysis identifies influential documents. Limitations include database biases, incomplete metadata, and search query specificity. The urgent need to explore genetic factors in cardiovascular diabetic neuropathy aligns with the increasing global diabetes burden. This analysis provides a comprehensive overview, contributing to the broader discourse on diabetic neuropathy research.

Range-wide genetic analysis of an endangered bumble bee (Bombus affinis, Hymenoptera: Apidae) reveals population structure, isolation by distance, and low colony abundance.

Declines in bumble bee species range and abundances are documented across multiple continents and have prompted the need for research to aid species recovery and conservation. The rusty patched bumble bee (Bombus affinis) is the first federally listed bumble bee species in North America. We conducted a range-wide population genetics study of B. affinis from across all extant conservation units to inform conservation efforts. To understand the species' vulnerability and help establish recovery targets, we examined population structure, patterns of genetic diversity, and population differentiation. Additionally, we conducted a site-level analysis of colony abundance to inform prioritizing areas for conservation, translocation, and other recovery actions. We find substantial evidence of population structuring along an east-to-west gradient. Putative populations show evidence of isolation by distance, high inbreeding coefficients, and a range-wide male diploidy rate of ~15%. Our results suggest the Appalachians represent a genetically distinct cluster with high levels of private alleles and substantial differentiation from the rest of the extant range. Site-level analyses suggest low colony abundance estimates for B. affinis compared to similar datasets of stable, co-occurring species. These results lend genetic support to trends from observational studies, suggesting that B. affinis has undergone a recent decline and exhibit substantial spatial structure. The low colony abundances observed here suggest caution in overinterpreting the stability of populations even where B. affinis is reliably detected interannually. These results help delineate informed management units, provide context for the potential risks of translocation programs, and help set clear recovery targets for this and other threatened bumble bee species.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays.

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

Clinical Characteristics and Surgical Outcomes of Patients Undergoing Pars Plana Vitrectomy for Proliferative Diabetic Retinopathy.

OBJECTIVE: To evaluate clinical characteristics impacting surgical outcomes of patients undergoing pars plana vitrectomy (PPV) for complications of proliferative diabetic retinopathy (PDR). DESIGN: Retrospective consecutive observational case series of patients at a large county hospital in Dallas, Texas, from 2014 to 2019. SUBJECTS: Seven hundred thirty-two patients (933 eyes) undergoing PPV for PDR complications. METHODS: Collected data included demographics, surgical indication, adjuvant therapies, intraoperative course, complications, and best corrected visual acuity (BCVA). Patients with < 6 months of follow-up were excluded. Best corrected visual acuity was converted to logarithm of the minimum angle of resolution for analysis. Statistics performed included t test, analysis of variance, and multivariate analyses. MAIN OUTCOME MEASURES: Postoperative BCVA, primary anatomic success rate, and postoperative complications. RESULTS: Three hundred ninety-three patients were male (509 eyes; 54.5%) with an average age of 52 years. Postoperative BCVA at 6 months was significantly different among surgical indications: 0.79 versus 0.77 versus 1.20 (P < 0.0001) for vitreous hemorrhage (VH), vitreomacular interface abnormalities, and tractional retinal detachment (TRD), respectively. Adjuvant preoperative therapy with panretinal photocoagulation (PRP) versus no PRP (0.95 vs. 1.25; P < 0.001) and insulin versus no insulin (0.99 vs. 1.17; P < 0.01) were associated with improved vision. Iatrogenic breaks were associated with decreased postoperative vision (1.40 vs. 0.88; P < 0.001). The primary anatomic success rate for TRD was 85% (495 eyes). Combined TRD/RRD (tractional and rhegmatogenous retinal detachment) was associated with a lower success rate compared with macula-on/macula-off TRD, with odds ratios of 0.36, 0.46, and 0.53, respectively. Patients experiencing recurrent detachment postsurgery had worse preoperative visual acuity (VA) (1.93 vs. 1.63; P < 0.01) and were younger (47.6 vs. 50.0; P = 0.02). Postoperative complications occurred in 699 eyes (75%), with VH (498 eyes, 53%), cataract (465, 50%), and elevated intraocular pressure (149, 16%) being the most common. Two hundred thirty-six eyes (25%) required a second PPV operation. Endophthalmitis (1 eye; <1%) and choroidal detachment (5 eyes; <1%) were rare. CONCLUSIONS: In this retrospective series analyzing surgical outcomes among patients with complications from PDR, vitrectomy led to improved vision on average, with a meaningful proportion of patients receiving additional surgical intervention. Surgical indication, presenting VA, age, and adjuvant therapies appeared to impact outcomes. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The alternative coproporphyrinogen III oxidase (CgoN) catalyzes the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III.

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in Bacillaceae, employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called ytpQ, encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from Priestia (Bacillus) megaterium. The recombinantly produced, purified, and monomeric YtpQ (CgoN) protein is shown to catalyze the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III. Minimal non-enzymatic conversion of coproporphyrinogen III was observed under the anaerobic test conditions employed in this study. FAD was identified as a cofactor, and menadione served as an artificial acceptor for the six abstracted electrons, with a KM value of 3.95 µmol/L and a kcat of 0.63 per min for the substrate. The resulting coproporphyrin III, in turn, acts as an effective substrate for the subsequent enzyme of the pathway, the coproporphyrin III ferrochelatase (CpfC). Under aerobic conditions, oxygen directly serves as an electron acceptor, but is replaced by the more efficient action of menadione. An AlphaFold2 model of the enzyme suggests that YtpQ adopts a compact triangular shape consisting of three domains. The N-terminal domain appears to be flexible with respect to the rest of the structure, potentially creating a ligand binding site that opens and closes during the catalytic cycle. A catalytic mechanism similar to the oxygen-independent protoporphyrinogen IX oxidase PgoH1 (HemG), based on the flavin-dependent abstraction of six electrons from coproporphyrinogen III and their potential quinone-dependent transfer to a membrane-localized electron transport chain, is proposed.

An Apple and Acáchul Berry Snack Rich in Bioaccessible Antioxidants and Folic Acid: A Healthy Alternative for Prenatal Diets.

A fruit leather (apple and acáchul berry) oriented toward women of reproductive age was developed. The snack was supplemented with an ingredient composed of folic acid (FA) and whey proteins (WPI) to ensure the required vitamin intake to prevent fetal neural tube defects. In order to generate a low-calorie snack, alternative sweeteners were used (stevia and maltitol). The fruit leather composition was determined. Also, an in vitro digestion process was carried out to evaluate the bioaccessibility of compounds with antioxidant capacity (AC), total polyphenols (TPCs), total monomeric anthocyanins (ACY), and FA. The quantification of FA was conducted by a microbiological method and by HPLC. The leather contained carbohydrates (70%) and antioxidant compounds, mainly from fruits. Bioaccessibility was high for AC (50%) and TPCs (90%), and low for ACY (17%). Regarding FA, bioaccessibility was higher for WPI-FA (50%) than for FA alone (37%), suggesting that WPI effectively protected the vitamin from processing and digestion. Furthermore, the product was shown to be non-cytotoxic in a Caco-2 cell model. The developed snack is an interesting option due to its low energy intake, no added sugar, and high content of bioactive compounds. Also, the supplementation with WPI-FA improved the conservation and bioaccessibility of FA.

Prevalence and Determinants of Plastic Surgery Among Adults in Saudi Arabia.

BACKGROUND: Social and cultural factors have a significant impact on body image standards, and the media's messages play a crucial role in shaping beauty ideals. People's perceptions of beauty can be negatively affected by social media, which promotes unrealistic beauty standards and can lead to a desire for plastic surgery. Plastic surgery includes both reconstructive and aesthetic procedures and has become increasingly popular worldwide. In Saudi Arabia, there has been a significant increase in the number of women seeking cosmetic surgery, particularly breast augmentation, rhinoplasty, and liposuction, indicating that cosmetic surgery is becoming more accepted as a way to enhance beauty. AIMS: To investigate the frequency of performance of plastic and aesthetic surgical procedures among adults aged 18 to 60 years old in Saudi Arabia, as well as the determinants that may contribute to it. METHODOLOGY: A cross-sectional web survey with a representative sample (n = 3238) of individuals in Saudi Arabia was carried out from 10 April 2023 to 28 October 2023. RESULTS: The study included a cohort of 3238 participants, with 1328 individuals choosing to undergo plastic surgery. Among the surgical subgroups, the predominant procedure of choice was breast augmentation, representing 1009 cases (31.2%), while a minority opted for alternative plastic surgical interventions. Notably, the primary impetus behind plastic surgery, as indicated by the majority of participants (38.4% of the overall 41% who underwent the procedure), was appearance enhancement. Examination of the participants' demographic profile revealed a predominance of women between the ages of 18 and 30, married individuals, predominantly having a primary level of education, with a subset working in the public sector. Furthermore, a considerable segment of participants (32.6%) indicated low income, while 31.8% fell into the category of obesity. CONCLUSION: To address the multifaceted nature of plastic surgery decisions in Saudi Arabia, the key focus should be on promoting cultural acceptance, creating a supportive environment, and establishing ethical guidelines. This entails conducting awareness campaigns, promoting inclusive beauty standards, facilitating community discussions, fostering collaboration with support organizations, integrating mental health services, and ensuring rigorous monitoring of safety and professionalism in plastic surgery practices. By implementing these recommendations, individuals seeking plastic surgery can enhance their overall well-being and satisfaction.

Cold-Inducible RNA Binding Protein Impedes Breast Tumor Growth in the PyMT Murine Model for Breast Cancer.

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Risk of Thromboembolism in Patients Receiving Immunotherapy-Based Combinations as Front-Line Therapy for Metastatic Renal Cell Carcinoma.

BACKGROUND: Most patients with treatment-naïve metastatic renal cell carcinoma (mRCC) receive combination-based immunotherapy with either 2 immune-oncology checkpoint inhibitors (IO/IO) or an IO agent in combination with a vascular endothelial growth factor receptor (VEGF-R) tyrosine kinase inhibitor (IO/TKI). The rates of thromboembolism (TE) in these cohorts are not clearly described and can potentially impact decision-making between IO/IO and IO/TKI. METHODS: We conducted a retrospective investigation of patients with treatment-naïve mRCC treated with IO-based combinations between January 2015 and April 2021 at the Cleveland Clinic. TE events, including venous and arterial, were identified in each group. Competing risk regression was done to identify factors associated with the development of TE following therapy, with all-cause mortality treated as a competing event. RESULTS: Of 180 patients identified, 77 (43%) received IO/TKI and 103 (57%) received IO/IO. Median age was 65 years, 75% were male, and 80% had clear cell histology. Baseline characteristics were similar between the 2 groups. At a median follow-up of 22.0 months, 10.0% of all patients had a TE. The one-year incidence of TE was 8.1% (95% CI: 3.3%-15.8%) with IO/TKI and 9.8% (95% CI: 5.0%-16.5%) with IO/IO and was not significantly different between the 2 groups (HR 0.89, 95% CI: 0.35%-2.28%). Occurrence of TE was associated with decreased overall survival regardless of IO/IO or IO/TKI therapy (HR 2.80, 95% CI: 1.57-5.02). There was no difference in incidence of TE based on patient age, gender, prior history of TE, International Metastatic Renal Cell Carcinoma (IMDC) risk group, or Khorana score. CONCLUSIONS: Incidence of TE is similar between IO/IO and IO/TKI regimens in treatment-naïve mRCC and is also associated with decreased overall survival. While risk of TE may not guide decision-making in choice of front-line mRCC therapy, careful attention should be given to the high risk of TE in this population.

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

Early Effects of Alpha-Synuclein Depletion by Pan-Neuronal Inactivation of Encoding Gene on Electroencephalogram Coherence between Different Brain Regions in Mice.

Inactivation of the Snca gene in young mice by chronic injections of tamoxifen (TAM), a selective estrogen receptor modifier, has been shown to decrease the level of alpha-synuclein, a key peptide in the pathogenesis of Parkinson's disease. In young mice, different time courses of the effect were observed in different brain areas, meaning associated disturbances in the intracerebral relations, namely in brain function after TAM-induced synucleinopathy. METHODS: We analyzed electroencephalogram (EEG) coherence ("functional connectivity") between the cortex (MC), putamen (Pt), and dopamine-producing brain regions (ventral tegmental area, VTA, and substantia nigra, SN) in two groups of two-month-old male mice. We compared EEG coherences in the conditional knockout Sncaflox/flox mice with those in their genetic background (C57Bl6J) one, two, and three months after chronic (for five days) intraperitoneal injections of TAM or the vehicle (corn oil). The EEG coherences in the TAM-treated group were compared with those in the alpha-synuclein knockout mice. RESULTS: A significant suppression of EEG coherence in the TAM-treated mice versus the vehicle group was observed in all inter-structural relations, with the exception of MC-VTA at one and three months and VTA-SN at two months after the injections. Suppressive changes in EEG coherence were observed in the alpha-synuclein knockout mice as well; the changes were similar to those in TAM-treated mice three months after treatment. CONCLUSION: our data demonstrate a combined time-dependent suppressive effect induced by TAM on intracerebral EEG coherence.

Distinct states of nucleolar stress induced by anticancer drugs.

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed 'nucleolar normality score' was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I- recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

Ribosomes are cell structures within a compartment called the nucleolus that are required to make proteins, which are essential for cell function. Due to their uncontrolled growth and division, cancer cells require many proteins and therefore have a particularly high demand for ribosomes. Due to this, some anti-cancer drugs deliberately target the activities of the nucleolus. However, it was not clear if anti-cancer drugs with other targets also disrupt the nucleolus, which may result in side effects. Previously, it had been difficult to study how nucleoli work, partly because in human cells they vary naturally in shape, size, and number. Potapova et al. used fluorescent microscopy to develop a new way of assessing nucleoli based on the location and ratio of certain proteins. These measurements were used to calculate a "nucleolar normality score". Potapova et al. then tested over a thousand anti-cancer drugs in healthy and cancerous human cells. Around 10% of the tested drugs changed the nucleolar normality score when compared to placebo treatment, indicating that they caused nucleolar stress. For most of these drugs, the nucleolus was not the intended target, suggesting that disrupting it was an unintended side effect. Drugs inhibiting proteins called cyclin-dependent kinases caused the most drastic changes in the size and shape of nucleoli, disrupting them completely. These kinases are known to be involved in activating enzymes required for general transcription. Potapova et al. showed that they also are involved in production of ribosomal RNA, revealing an additional role in coordinating ribosome assembly. Taken together, the findings suggest that evaluating the effect of new anti-cancer drugs on the nucleolus could help to develop future treatments with less toxic side effects. The experiments also reveal new avenues for researching how cyclin-dependent kinases control the production of RNA more generally.