An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination.

Polyadenylation is a co-transcriptional process that modifies mRNA 3'-ends in eukaryotes. In yeast, CF IA and CPF constitute the core 3'-end maturation complex. CF IA comprises Rna14p, Rna15p, Pcf11p and Clp1p. CF IA interacts with the C-terminal domain of RNA Pol II largest subunit via Pcf11p which links pre-mRNA 3'-end processing to transcription termination. Here, we analysed the role of Clp1p in 3' processing. Clp1p binds ATP and interacts in CF IA with Pcf11p only. Depletion of Clp1p abolishes transcription termination. Moreover, we found that association of mutations in the ATP-binding domain and in the distant Pcf11p-binding region impair 3'-end processing. Strikingly, these mutations prevent not only Clp1p-Pcf11p interaction but also association of Pcf11p with Rna14p-Rna15p. ChIP experiments showed that Rna15p cross-linking to the 3'-end of a protein-coding gene is perturbed by these mutations whereas Pcf11p is only partially affected. Our study reveals an essential role of Clp1p in CF IA organization. We postulate that Clp1p transmits conformational changes to RNA Pol II through Pcf11p to couple transcription termination and 3'-end processing. These rearrangements likely rely on the correct orientation of ATP within Clp1p.

Nodular image in the appendix observed on ultrasound: endometriosis or neuroendocrine neoplasia?

Objective: To evaluate the association between clinical and imaging with surgical and pathological findings in patients with suspected neuroendocrine tumor of appendix and/or appendix endometriosis. Methods: Retrospective descriptive study conducted at the Teaching and Research Institute of Hospital Israelita Albert Einstein, in which medical records and databases of patients with suspected neuroendocrine tumor of appendix and/or endometriosis of appendix were analyzed by imaging. Results: Twenty-eight patients were included, all of which had some type of appendix alteration on the ultrasound examination. The pathological outcome of the appendix found 25 (89.3%) lesions compatible with endometriosis and three (10.7%) neuroendocrine tumors. The clinical findings of imaging and surgery were compared with the result of pathological anatomy by means of relative frequency. Conclusion: It was possible to observe a higher prevalence of appendix endometriosis when the patient presented more intense pain symptoms. The image observed on ultrasound obtained a high positive predictive value for appendicular endometriosis.

Developing a Nomogram for Prioritizing Hysteroscopy in Endometrial Cancer Diagnosis: A Case-Control Study.

(1) Background: The pandemic led to significant healthcare disruptions, resulting in postponed surgeries and extended waiting times for non-urgent treatments, including hysteroscopies essential for diagnosing endometrial cancer. This study aims to formulate a risk stratification model to enhance the prioritization of hysteroscopy procedures in Brazil; (2) Methods: A case-control study was conducted at Vila Santa Catarina Hospital in São Paulo, analyzing the medical records of 2103 women who underwent hysteroscopy between March 2019 and March 2022. We used bivariate analysis and multivariate linear regression to identify risk factors associated with endometrial cancer and formulate a nomogram; (3) Results: The findings revealed a 5.5% incidence of pre-invasive and invasive endometrial disease in the study population, with an average waiting time of 120 days for hysteroscopy procedures. The main risk factors identified were hypertension, diabetes, postmenopausal bleeding, and obesity; (4) Conclusions: This research highlights the urgent need for efficient prioritization of hysteroscopy procedures in the wake of the pandemic. The developed nomogram is an innovative tool for identifying patients at higher risk of endometrial cancer, thus facilitating timely diagnosis and treatment and improving overall patient outcomes in a strained healthcare system.

Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange.

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.

Nodular image in the appendix observed on ultrasound: endometriosis or neuroendocrine neoplasia?

Abstract Objective: To evaluate the association between clinical and imaging with surgical and pathological findings in patients with suspected neuroendocrine tumor of appendix and/or appendix endometriosis. Methods: Retrospective descriptive study conducted at the Teaching and Research Institute of Hospital Israelita Albert Einstein, in which medical records and databases of patients with suspected neuroendocrine tumor of appendix and/or endometriosis of appendix were analyzed by imaging. Results: Twenty-eight patients were included, all of which had some type of appendix alteration on the ultrasound examination. The pathological outcome of the appendix found 25 (89.3%) lesions compatible with endometriosis and three (10.7%) neuroendocrine tumors. The clinical findings of imaging and surgery were compared with the result of pathological anatomy by means of relative frequency. Conclusion: It was possible to observe a higher prevalence of appendix endometriosis when the patient presented more intense pain symptoms. The image observed on ultrasound obtained a high positive predictive value for appendicular endometriosis.

Preventing Radiation-Induced Injury by Topical Application of an Amifostine Metabolite-Loaded Thermogel.

PURPOSE: Despite the development of high-precision radiation therapy, ionizing radiation inevitably damages healthy tissues. Radiodermatitis and radioinduced oral mucositis are frequent and significant side effects among patients with breast and head and neck cancer, respectively. These radiation-related injuries negatively affect patient quality of life and can lead to unplanned therapeutic breaks and compromise treatment outcomes. Currently, no preventive or mitigating agent has emerged to address these issues. Although amifostine, a well-known free radical scavenger, has proven efficacy against specific radio- and chemo-induced toxicities, severe adverse side effects (reversible hypotension, nausea, emesis, etc) combined with logistical hurdles are associated with its recommended intravenous route of administration, limiting its use. METHODS AND MATERIALS: We developed a thermogel containing the active thiol metabolite of amifostine (CPh-1014) that polymerizes at body temperature and serves as a matrix for topical application onto the skin or mucosa. RESULTS: Applied before irradiation, CPh-1014 greatly reduced the severity of oral mucositis and dermatitis induced by either a single dose or fractionated irradiation regimens in in vivo mouse models. The cytoprotective effect of CPh-1014 was confirmed by the decrease in DNA double-strand breaks in the irradiated epithelium. Noticeably, CPh-1014 did not affect radiation therapy efficacy against tumors grafted at submucosal and subcutaneous sites. In contrast to the intravenous administration of amifostine, CPh-1014 oral application did not induce hypotension in dogs. CONCLUSIONS: CPh-1014 confers radioprotective effects in healthy tissues with reduced systemic side effects without compromising radiation therapy efficacy. We propose CPh-1014 as an easy-to-implement therapeutic approach to alleviate radiation therapy toxicity in patients with breast and head and neck cancer.

Association between col1a2 Polymorphism and the Occurrence of Pelvic Organ Prolapse in Brazilian Women.

OBJECTIVE: To evaluate the rs42524 polymorphism of the procollagen type I alpha (α) 2 (COL1A2) gene as a factor related to the development of pelvic organ prolapse (POP) in Brazilian women. METHODS: The present study involved 112 women with POP stages III and IV (case group) and 180 women with POP stages zero and I (control group). Other clinical data were obtained by interviewing the patients about their medical history, and blood was also collected from the volunteers for the extraction of genomic DNA. The promoter region of the COL1A2 gene containing the rs42524 polymorphism was amplified, and the discrimination between the G and C variants was performed by digestion of the polymerase chain reaction (PCR) products with the MspA1I enzyme followed by agarose gel electrophoresis analysis. RESULTS: A total of 292 women were analyzed. In the case group, 71 had the G/G genotype, 33 had the G/C genotype, and 7 had the C/C genotype. In turn, the ratio in the control group was 117 G/G, 51 G/C, and 11 C/C. There were no significant differences between the groups. CONCLUSION: Our data did not show an association between the COL1A2 polymorphism and the occurrence of POP.

OBJETIVO: Avaliar o polimorfismo rs42524 do gene pró-colágeno tipo I alfa (α) 2 (COL1A2) como fator relacionado ao desenvolvimento de prolapso de órgãos pélvicos (POP) em mulheres brasileiras. MéTODOS: O estudo envolveu 112 mulheres com POP nos estádios III e IV (grupo caso) e 180 mulheres com POP nos estádios zero e I (grupo controle). Outros dados clínicos foram obtidos por meio de entrevistas com as pacientes sobre seu histórico médico, e o sangue das voluntárias também foi coletado para extração de DNA genômico. A região promotora do gene COL1A2 contendo o polimorfismo rs42524 foi amplificada, e a discriminação entre as variantes G e C foi realizada por digestão dos produtos de reação em cadeia da polimerase (RCP) com a enzima MspA1I, seguida de análise por eletroforese em gel de agarose. RESULTADOS: Foram analisadas 292 mulheres. No grupo caso, 71 tinham o genótipo G/G, 33 tinham o genótipo G/C, e 7 tinham o genótipo C/C. Por sua vez, a relação no grupo controle foi de 117 G/G, 51 G/C e 11 C/C. Não houve diferenças significativas entre os grupos. CONCLUSãO: Nossos dados não mostraram associação do polimorfismo do gene COL1A2 com a ocorrência de POP.

Naftazone in advanced Parkinson's disease: An acute L-DOPA challenge randomized controlled trial.

INTRODUCTION: There is an unmet need to better control motor complications in Parkinson's disease (PD). Naftazone, which exhibits glutamate release inhibition properties, has shown antiparkinsonian and antidyskinetic activity in preclinical models of PD and in a clinical proof of concept study. METHODS: We conducted a double-blind randomized placebo-controlled cross-over trial in PD patients with motor fluctuations and dyskinesia testing naftazone 160 mg/day versus placebo for 14 days. The two co-primary endpoints were the area under curve (AUC) of motor (MDS-UPDRS part III) and dyskinesia (AIMS) scores during an acute levodopa challenge performed at the end of each period. Secondary endpoints were UDysRS and axial symptoms scores during the challenge; AIMS, UDysRS, and time spent with or without dyskinesia the day before the challenge. The primary analysis was performed in the per protocol population. RESULTS: Sixteen patients were included in the analysis. There was no difference between naftazone and placebo for the AUC of MDS-UPDRS III (-89, 95%CI[-1071; 893], p = 0.85), and AIMS (70, 95%CI[-192; 332], p = 0.57). At the end of treatment periods, AIMS score tended to be lower with naftazone than placebo (4.4 ±â€¯3.4 versus 6.7 ±â€¯4.4, p = 0.07), but UDysRS scores and other secondary outcomes were not different. Naftazone was safe and well tolerated. CONCLUSIONS: This study did not confirm previous results on the efficacy of naftazone on dyskinesia nor motor fluctuations highlighting the problem of translating results obtained in preclinical models into clinical trials. Further investigation of naftazone may be conducted in PD with longer treatment duration.

Purification of FLAG-tagged eukaryotic initiation factor 2B complexes, subcomplexes, and fragments from Saccharomyces cerevisiae.

The eukaryotic initiation factor 2B (eIF2B) is a five-subunit guanine nucleotide exchange factor, that functions during translation initiation to catalyze the otherwise slow exchange of GDP for GTP on its substrate eIF2. Assays to measure substrate interaction and guanine nucleotide release ability of eIF2B require the complex to be purified free of interacting proteins. We have also found that a subcomplex of two subunits, gamma and epsilon or the largest one, epsilon alone, promotes this activity. Within eIF2Bepsilon, the catalytic center requires the C-terminal 200 residues only. Here, we describe our protocols for purifying the Saccharomyces cerevisiae eIF2B complexes and the catalytic subunit using FLAG-tagged proteins overexpressed in yeast cells. Using commercially available FLAG-affinity resin and high salt buffer, we are able to purify active eIF2B virtually free of contaminants.

Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication.

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.

Association between col1a2 polymorphism and the occurrence of pelvic organ prolapse in Brazilian women / Associação entre polimorfismo do gene col1a2 e a ocorrência do prolapso de órgãos pélvicos em mulheres brasileiras

Abstract Objective To evaluate the rs42524 polymorphism of the procollagen type I alpha (α) 2 (COL1A2) gene as a factor related to the development of pelvic organ prolapse (POP) in Brazilian women. Methods The present study involved 112 women with POP stages III and IV (case group) and 180 women with POP stages zero and I (control group). Other clinical data were obtained by interviewing the patients about their medical history, and blood was also collected from the volunteers for the extraction of genomic DNA. The promoter region of the COL1A2 gene containing the rs42524 polymorphism was amplified, and the discrimination between the G and C variants was performed by digestion of the polymerase chain reaction (PCR) products with the MspA1I enzyme followed by agarose gel electrophoresis analysis. Results A total of 292 women were analyzed. In the case group, 71 had the G/G genotype, 33 had the G/C genotype, and 7 had the C/C genotype. In turn, the ratio in the control group was 117 G/G, 51 G/C, and 11 C/C. There were no significant differences between the groups. Conclusion Our data did not show an association between the COL1A2 polymorphism and the occurrence of POP.

Resumo Objetivo Avaliar o polimorfismo rs42524 do gene pró-colágeno tipo I alfa (α) 2 (COL1A2) como fator relacionado ao desenvolvimento de prolapso de órgãos pélvicos (POP) em mulheres brasileiras. Métodos O estudo envolveu 112 mulheres com POP nos estádios III e IV (grupo caso) e 180 mulheres com POP nos estádios zero e I (grupo controle). Outros dados clínicos foramobtidos pormeio de entrevistas comas pacientes sobre seu históricomédico, e o sangue das voluntárias também foi coletado para extração de DNA genômico. A região promotora do gene COL1A2 contendo o polimorfismo rs42524 foi amplificada, e a discriminação entre as variantes G e C foi realizada por digestão dos produtos de reação em cadeia da polimerase (RCP) com a enzima MspA1I, seguida de análise por eletroforese em gel de agarose. Resultados Foram analisadas 292 mulheres. No grupo caso, 71 tinham o genótipo G/G, 33 tinham o genótipo G/C, e 7 tinham o genótipo C/C. Por sua vez, a relação no grupo controle foi de 117 G/G, 51 G/C e 11 C/C. Não houve diferenças significativas entre os grupos. Conclusão Nossos dados não mostraram associação do polimorfismo do gene COL1A2 com a ocorrência de POP.

Blood transcriptomic biomarkers of Alzheimer's disease patients treated with EHT 0202.

Monitoring the genomic expression of patients in clinical trials for Alzheimer's disease (AD) can assist trial design and treatment response analysis. Here, we report on the identification in AD patients of blood-based transcriptomic signatures associated with treatment response of EHT 0202, a new compound with potential disease-modifying and symptomatic properties, in a 3-month, placebo-controlled, Phase IIA study aimed at determining the clinical safety, tolerability, and exploratory efficacy of EHT 0202 (40 and 80 mg bid) as adjunctive therapy to one cholinesterase inhibitor in mild to moderate AD patients. Genome-wide transcriptomic profiling was performed on blood samples taken prior to treatment and at study completion in a subpopulation of 60 AD patients selected as either the 10 worst disease decliners or the 10 best improvers of each treatment group, using ADAS-Cog scores as measure of disease severity. In the patients responding to EHT 0202, a pre-treatment (baseline) transcriptomic signature showed activation of pathways related to AD, CNS disorders, diabetes, inflammation, and autoimmunity, while a post-treatment signature indicated reduced activation of these pathways with induced metabolic and transcription stimulation. This pilot study demonstrates the utility of blood transcriptomic signatures used as biomarkers for predicting patient response or monitoring efficacy, for an administered therapeutic drug in a complex disease such as AD. For EHT 0202 or other AD drugs, such biomarkers may help to improve strategies to better identify appropriate patient populations for treatment, understand the drug mechanism of efficacy, and/or clarify the inherent subjectivity in most clinical endpoints used in this disease.

Validation of AclarusDx™, a blood-based transcriptomic signature for the diagnosis of Alzheimer's disease.

Biomarkers have gained an increased importance in the past years in helping physicians to diagnose Alzheimer's disease (AD). This study was designed to identify a blood-based, transcriptomic signature that can differentiate AD patients from control subjects. The performance of the signature was then evaluated for robustness in an independent blinded sample population. RNA was extracted from 177 blood samples (90 AD patients and 87 controls) and gene expression profiles were generated using the human Genome-Wide Splice Array™. These profiles were used to establish a signature to differentiate AD patients from controls. Subsequently, prediction results were optimized by establishing grey zone boundaries that discount prediction scores near the disease status threshold. Signature validation was then performed on a blinded independent cohort of 209 individuals (111 AD and 98 controls). The AclarusDx™ signature consists of 170 probesets which map to 136 annotated genes, a significant number of which are associated with inflammatory, gene expression, and cell death pathways. Additional signature genes are known to interact with pathways involved in amyloid and tau metabolism. The validation sample set, after removal of 45 individuals with prediction profile scores within the grey zone, consisted of 164 subjects. The AclarusDx™ performance on this validation cohort had a sensitivity of 81.3% (95% CI: [73.3%; 89.3%]); and a specificity of 67.1% (95% CI: [56.3%; 77.9%]). AclarusDx™ is a non-invasive blood-based transcriptomic test that, in combination with standard assessments, can provide physicians with objective information to support the diagnosis of AD.