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Pancreatic neuroendocrine tumors (PNETs) are malignancies arising from the islets of Langerhans. Therapeutic options are limited for the over 50% of patients who present with metastatic disease. We aimed to identify mechanisms to remodel the PNET tumor microenvironment (TME) to ultimately enhance susceptibility to immunotherapy. The TMEs of localized and metastatic PNETs were investigated using an approach that combines RNA-Seq, cancer and T cell profiling, and pharmacologic perturbations. RNA-Seq analysis indicated that the primary tumors of metastatic PNETs showed significant activation of inflammatory and immune-related pathways. We determined that metastatic PNETs featured increased numbers of tumor-infiltrating T cells compared with localized tumors. T cells isolated from both localized and metastatic PNETs showed evidence of recruitment and antigen-dependent activation, suggestive of an immune-permissive microenvironment. A computational analysis suggested that vorinostat, a histone deacetylase inhibitor, may perturb the transcriptomic signature of metastatic PNETs. Treatment of PNET cell lines with vorinostat increased chemokine CCR5 expression by NF-κB activation. Vorinostat treatment of patient-derived metastatic PNET tissues augmented recruitment of autologous T cells, and this augmentation was substantiated in a mouse model of PNET. Pharmacologic induction of chemokine expression may represent a promising approach for enhancing the immunogenicity of metastatic PNET TMEs.

Abstract During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients. Here, two human cell models were generated to investigate how a mutation in the C-terminal low-complexity domain (LCD) of hnRNPA1 can cause splicing changes of thousands of transcripts that collectively are linked to the DNA damage response, cilium organization, and translation. We show that the hnRNPA1 D262V mutant protein binds to new binding sites on differentially spliced transcripts from genes that are linked to ALS. We demonstrate that this ALS-linked hnRNPA1 mutation alters normal RNA-dependent protein-protein interactions. Furthermore, cells expressing this hnRNPA1 mutant exhibit a cell aggregation phenotype, markedly reduced growth rates, changes in stress granule kinetics, and aberrant growth of neuronal processes. This study provides insight into how a single amino acid mutation in a splicing factor can alter RNA splicing networks of genes linked to ALS.

Regulation of messenger RNA (mRNA) stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV) promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A) polymerase II-generated poly(A) tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A) tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A) binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A) tail-stimulated mRNA turnover in mammalian cells.

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

Context The significance of low mitotic activity in papillary thyroid cancer (PTC) is largely undefined. Objective We aimed to determine the behavioral landscape of PTC with low mitotic activity compared with that of no and high mitotic activity. Methods A single-institution consecutive series of patients with PTC from 2018 to 2022 was reviewed. Mitotic activity was defined as no mitoses, low (1-2 mitoses/2 mm2) or high (≥3 mitoses/2 mm2) per the World Health Organization. The 2015 American Thyroid Association risk stratification was applied to the cohort, and clinicopathologic features were compared between groups. For patients with ≥6 months of follow-up, Cox regression analyses for recurrence were performed. Results A total of 640 PTCs were included—515 (80.5%) no mitotic activity, 110 (17.2%) low mitotic activity, and 15 (2.3%) high mitotic activity. Overall, low mitotic activity exhibited rates of clinicopathologic features including vascular invasion, gross extrathyroidal extension, and lymph node metastases in between those of no and high mitotic activity. PTCs with low mitotic activity had higher rates of intermediate- and high-risk American Thyroid Association (ATA) risk stratification compared with those with no mitotic activity (P <.001). Low mitotic activity PTCs also had higher recurrence rates (15.5% vs 4.5%, P <.001). Low mitotic activity was associated with recurrence, independent of the ATA risk stratification (HR 2.96; 95% CI 1.28-6.87, P =.01). Conclusion Low mitotic activity is relatively common in PTC and its behavior lies within a spectrum between no and high mitotic activity. Given its association with aggressive clinicopathologic features and recurrence, low mitotic activity should be considered when risk stratifying patients with PTC for recurrence. [ABSTRACT FROM AUTHOR]

Context Pancreatic neuroendocrine tumors (PNETs) exhibit a wide range of behavior from localized disease to aggressive metastasis. A comprehensive transcriptomic profile capable of differentiating between these phenotypes remains elusive. Objective Use machine learning to develop predictive models of PNET metastatic potential dependent upon transcriptomic signature. Methods RNA-sequencing data were analyzed from 95 surgically resected primary PNETs in an international cohort. Two cohorts were generated with equally balanced metastatic PNET composition. Machine learning was used to create predictive models distinguishing between localized and metastatic tumors. Models were validated on an independent cohort of 29 formalin-fixed, paraffin-embedded samples using NanoString nCounter®, a clinically available mRNA quantification platform. Results Gene expression analysis identified concordant differentially expressed genes between the 2 cohorts. Gene set enrichment analysis identified additional genes that contributed to enriched biologic pathways in metastatic PNETs. Expression values for these genes were combined with an additional 7 genes known to contribute to PNET oncogenesis and prognosis, including ARX and PDX1. Eight specific genes (AURKA, CDCA8, CPB2, MYT1L, NDC80, PAPPA2, SFMBT1, ZPLD1) were identified as sufficient to classify the metastatic status with high sensitivity (87.5-93.8%) and specificity (78.1-96.9%). These models remained predictive of the metastatic phenotype using NanoString nCounter® on the independent validation cohort, achieving a median area under the receiving operating characteristic curve of 0.886. Conclusion We identified and validated an 8-gene panel predictive of the metastatic phenotype in PNETs, which can be detected using the clinically available NanoString nCounter® system. This panel should be studied prospectively to determine its utility in guiding operative vs nonoperative management. [ABSTRACT FROM AUTHOR]

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein-RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.

Neopterin may be relevant for colorectal cancer (CRC) development, as a biomarker of cellular immune activity exerting pleiotropic effects on cellular ageing, oxidative stress, and inflammation. So far, the association between prediagnostic neopterin and colon and rectal cancer risk has not been evaluated in human populations. A nested case-control study was conducted within the European Prospective Investigation into Cancer and Nutrition cohort using data on plasma concentrations of total neopterin (T-N, sum of neopterin and 7,8-dihydroneopterin) in 830 incident CRC case patients (561 colon and 269 rectal) matched within risk sets to 830 control participants. A subsequent replication study used data from the Hordaland Health Study, where 173 CRC case patients have been diagnosed among 6594 healthy participants over 12 years of follow-up. After multivariable adjustment for a priori chosen CRC risk factors, a "U-shaped" association of T-N with CRC was revealed. Compared with the second quintile of the T-N distribution, the relative risks for the first, third, fourth, and fifth quintiles were 2.37 (95% CI = 1.66 to 3.39), 1.24 (95% CI = 0.87 to 1.77), 1.55 (95% CI = 1.08 to 2.22), and 2.31 (95% CI = 1.63 to 3.27), respectively. Replication of these associations within the Hordaland Health Study yielded similar results. No differences have been observed when the associations were explored by colon and rectal cancer site (two-sided P difference = .87) and after excluding case patients diagnosed within the first four follow-up years. These novel findings provide evidence of the role of both suppressed and activated cell-mediated immunity as reflected by prediagnostic T-N concentrations in the development of CRC.

Regulation of messenger RNA (mRNA) stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV) promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A) polymerase II-generated poly(A) tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A) tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A) binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A) tail-stimulated mRNA turnover in mammalian cells.

Abstract Over 75% of failed pregnancies involve implantation defects. Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein involved in signal transduction and cell communication. Here we show that the expression of GRB2 protein is lower in endometrium of infertile women with endometriosis compared to controls. Our mouse endometriosis model revealed that endometriosis development results to GRB2 loss in the eutopic endometrium. To understand the role of GRB2 in the uterus, we generated mice with conditional ablation of Grb2 in the Pgr positive cells (Grb2 d/d ). Grb2 d/d mice were infertile due to implantation failure. Although ovarian functions were normal, Grb2 d/d mice had a non-receptive endometrium due to progesterone resistance and dysregulation of steroid hormone and FOXA2 signaling pathways. Furthermore, our results were supported by findings of GRB2 attenuation in primary human endometrial stromal cells from women with endometriosis. Our results demonstrate that GRB2 is critical for endometrial receptivity and decidualization.

Purpose: This study aimed to report the results of Korean Antimicrobial Resistance Monitoring System (KARMS) for uncomplicated cystitis (UC) in 2023. Materials and Methods: KARMS was established for the surveillance of antimicrobial resistance in urinary tract infections with the cooperation of Korean nationwide medical centers. Data from patients with UC have been collected in the web-based KARMS database. Demographic data, uropathogen distribution, and antimicrobial susceptibility of representative pathogens were analyzed. Results: A total of 885 patients' data were collected in KARMS database. The mean patient age was 56.39±18.26 years. The number of postmenopausal and recurrent cystitis were 530 (61.1%) and 102 (11.5%), respectively. Escherichia coli was the most frequently identified uropathogen (654/871, 75.1%). Regarding antimicrobial susceptibility, 94.9% were susceptible to fosfomycin, 90.5% to nitrofurantoin, 58.4% to ciprofloxacin, 83.6% to cefotaxime, and 100.0% to ertapenem. ESBL positivity was 13.7% (96/702), and significantly higher in tertiary hospital (23.1%, p

Background Residents and nurses who activate rapid response teams (RRTs) are well positioned to offer insights on its effectiveness. Here, we assess such evaluation of RRTs and identify barriers to activation in a 1,400-bed teaching hospital. Methods We conducted a 24-item Likert-scale survey from January to May 2017 among residents and ward nurses with RRT experience. Factor analysis was used to identify the barriers. Results This study comprised 305 nurses and 53 residents, most of whom were satisfied with their RRT experiences. Factor analysis showed that lack of awareness of activation criteria was a major barrier, with only 21.4% and 22.2% participants, respectively, confident about their knowledge of activation protocols. Of the survey respondents, 85.7% reported first contacting the doctor before activating the RRT. Despite the protocol, 66.7% first discussed the decision with other staff, and 71.5% called the RRT when the patient’s condition worsened despite management. Conclusions Nurses and residents value RRTs but face barriers in initiation, primarily due to a lack of confidence in applying the activation criteria. Many prefer to consult a doctor or manage the patient before calling the RRT.

Abstract Background Early remdesivir administration in high-risk patients with coronavirus disease 2019 (COVID-19) is known to be effective in preventing the progression to severe disease. However, the effect of early remdesivir administration on Omicron variants, which are known to have decreased severity, remains unclear. Methods This study aimed to analyze the effects of early remdesivir administration during the Omicron wave in hospitalized patients. Electronic medical records of hospitalized patients with confirmed COVID-19 between February 2022 and February 2023 were reviewed. We included patients aged ≥ 18 years who had symptom onset within 7 days and had at least one risk factor for disease progression at the time of diagnosis. We compared the clinical outcomes between the early remdesivir administration group and the group not administered early remdesivir. The primary outcome was all-cause mortality within 28 days and the secondary outcome was the need for oxygen supplementation within 28 days. Multivariable analysis was conducted to assess risk factors for all-cause mortality and the need for oxygen supplementation. Results A total of 286 patients were enrolled, including 88 in the early remdesivir administration group and 198 in the control group. Clinical outcomes, including all-cause mortality (3.4% vs. 6.1%, P = 0.556) and need for oxygen supplementation (15.9% vs. 14.6%, P = 0.783) within 28 days, were not significantly different between the two groups. Age (HR, 1.061; 95% CI: 1.002, 1.124; P = 0.043), BMI (HR, 0.849; 95% CI: 0.725, 0.994; P = 0.041), and malignancy (HR, 4.619; 95% CI: 1.618, 13.189; P = 0.004) were identified as independent factors associated with all-cause mortality. Additionally, BMI (OR, 0.908; 95% CI, 0.824, 1.000; P = 0.049) and vaccination with more than three doses (OR, 0.412; 95% CI, 0.202, 0.839; P = 0.015) were independent factors associated with the need for oxygen supply. Early remdesivir administration was not significantly associated with all-cause mortality (HR, 0.393; 95% CI: 0.109, 1.417; P = 0.154) or the need for oxygen supplementation (OR, 0.823; 95% CI: 0.389, 1.740; P = 0.610). Conclusions In our study, early remdesivir administration was not associated with preventing progression to severe disease when used as previously indicated during the Omicron wave. Considering the decline in the severity of the Omicron variant and the increased vaccination rate reported in previous studies, further studies are needed to establish new indications for the use of early remdesivir in the Omicron variant.

The genus Campylomyza Meigen, 1818, belongs to the subfamily Micromyinae (Diptera, Cecidomyiidae). The genus, comprising 40 species, is best known in the Palearctic Region. To date, four species are recorded in Korea: Campylomyza appendiculata, C. flavipes, C. furva, and C. spinata. Based on our field investigations from 2017 to 2020, five species are newly recorded from Korea (C. abjecta, C. aborigena, C. cornuta, C. cavitata, and C. cingulata) and seven new species are described (C. ambulata sp. nov., C. angusta sp. nov., C. convexa sp. nov., C. cornigera sp. nov., C. hori sp. nov., C. odae sp. nov., and C. salicia sp. nov.), based on morphological identification and molecular analyses. Detailed morphological and molecular data, including mitochondrial COI sequences are provided, with species diagnosis, descriptions, and keys for identification of those species.

Abstract Retained medullary cord (RMC) is a recently defined term denoting closed spinal dysraphism arising from the failure of regression in secondary neurulation. Despite the acknowledgment of this condition, there needs to be more literature elucidating the radiologic manifestations of RMC. This study aimed to describe the MR imaging findings of RMC. A retrospective analysis was conducted on spinal MRI scans of pediatric patients with surgically confirmed RMC, from January 2014 to August 2023. The investigation focused on evaluating the following image characteristics: level and morphology of the cord-like structure (C-LS), the signal intensity in the far distal C-LS, nerve root-like structures originating from the C-LS, intradural fatty lesions, and arachnoid cysts. This study included 38 patients (19 girls, mean age 7.3 months) who showed a low-lying cord in all cases. The morphology of C-LS was either smooth tapered (50%) or hourglass with fusiform cystic dilatation (50%). The C-LS exhibited aberrant T2 hypointense signal compared to the juxta-proximal level. T2 hypointense signal vestigial nerves emanating from the C-LS and intradural fatty masses were observed (89.5%). Sacral arachnoid cysts in extradural location were identified in eight patients. The characteristic MR features of RMC revealed an extremely low-lying distal C-LS with smooth tapering or hourglass-shaped cystic dilatation of the caudal part, accompanied by an intradural fatty stalk and the aberrant signal vestigial nerve. This study suggests those radiologic findings can be RMC instead of the previously called terminal syrinx with low-lying conus.

Soybean [Glycine max (L.)] has higher nitrogen requirements than other crops. We investigated the effects on soybean yield and quality of topdressing with nitrogen fertilizer. Nitrogen fertilizer was applied as a topdressing to soybeans at 0, 20, 30, and 40 kg ha−1 (N0, N20, N30, and N40 treatments, respectively); half of the total topdressing treatment was applied at the pre-flowering (R 1) stage and the other half at the post-flowering (R 2) stage. Yield was highest in the N20 treatment and decreased with larger quantities of topdressing. The protein and total amino acid content were highest in the N20 treatment but tended to decrease with a greater quantity of topdressing. contents of most individual amino acids peaked in the N20 or N30 treatments and decreased as topdressing quantity increased, although proline and arginine contents increased with quantity of topdressing. Isoflavone content tended to be highest in either the N30 or N20 treatment. The agronomic efficiency of nitrogen (AEN) was highest in the N20 treatment. There was a positive correlation between AEN and yield, protein, isoflavone, and amino acid content. Topdressing with 20 kg ha−1 N produced the highest yield, protein, and amino acid content. Topdressing with greater quantities of nitrogen fertilizer decreased the yield and quality of soybeans.

Probiotics are thought to be effective in the treatment of radiation-induced enteropathy (RIE). However, little is known regarding their efficacy in preventing RIE. In this prospective, randomized, double-blinded, placebo-controlled, single-center study, the incidence of grade 2 acute RIE was compared and the safety of probiotics was evaluated. Patients receiving pelvic radiotherapy for a minimum of 40 Gy at the pelvic level were randomized into two groups: (i) a probiotic group receiving Bacillus licheniformis from two weeks before radiotherapy until the end and (ii) a control group receiving a placebo with the same schedule. The toxicities of 234 patients were graded according to the Common Terminology Criteria for Adverse Events v5.0. Grade 1 RIE was observed in 65 (56%) of the probiotics group compared with 75 (64%) of the placebo group. Grade 2 RIE occurred in 30 patients (26%) in the probiotics group compared with 26 (22%) in the placebo group, indicating that probiotics failed in their preventive role compared with placebo (p = 0.493). Medication adherence rates were good, and no difference was observed between the two arms. These findings suggest that B. licheniformis does not significantly prevent RIE.

Abstract Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity.

Abstract Background The unprecedented pandemic situation of COVID-19 has had a negative impact on the mental health of many people, especially among the "old-old" older adults who are aged 75 or older. Therefore, the aim of this study is to investigate the changes in depression among "old-old" older adults before and after the onset of COVID-19, and the extent to which depression affects suicidal ideations. Method The 12th to 16th Korea Welfare Panel Study(KoWePS) conducted from 2017 to 2021 was used for analysis. For this study, 771 older individuals with complete data to estimate the degree of change of depression were selected as the final analysis subjects. Result A Growth Mixture Modeling(GMM) analysis was conducted, resulting in the classification of two groups: an increasing group and a decreasing group. The study findings showed that "old-old" older individuals with high levels of depression, specifically those in the decreasing group, may be more susceptible to suicidal ideation. Despite this steep change slope, the decreasing group still exhibited a higher level of depression in 2021 compared to the increasing group. As per characteristics, the decreasing group, which showed a higher prevalence of suicidal ideation, had a higher proportion of women and individuals with lower levels of education, those living alone, and a lower household income compared to the increasing group. Conclusion It is important to note that although the study emphasized the need to prioritize intervention for the decreasing group with consistently high levels of depression, the majority of individuals belong to the increasing group, which exhibited a gradual increase in depression levels over time. Therefore, intervention plans should be developed concurrently for both groups. Also, it is crucial to implement proactive efforts targeting groups with understandings of these characteristics when establishing preventative measures for depression and suicidal ideation among "old-old" older adults.