Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Anti-tumor efficacy assessment of the sigma receptor pan modulator RC-106. A promising therapeutic tool for pancreatic cancer

Introduction: Pancreatic cancer (PC) is one of the most lethal tumor worldwide, with no prognosis improvement over the past 20-years. The silent progressive nature of this neoplasia hampers the early diagnosis, and the surgical resection of the tumor, thus chemotherapy remains the only available therapeutic option. Sigma receptors (SRs) are a class of receptors proposed as new cancer therapeutic targets due to their over-expression in tumor cells and their involvement in cancer biology. The main localization of these receptors strongly suggests their potential role in ER unfolded protein response (ER-UPR), a condition frequently occurring in several pathological settings, including cancer. Our group has recently identified RC-106, a novel pan-SR modulator with good in vitro antiproliferative activities toward a panel of different cancer cell lines. In the present study, we investigated the in vitro properties and pharmacological profile of RC-106 in PC cell lines with the aim to identify a potential lead candidate for the treatment of this tumor. Methods: Pancreatic cancer cell lines Panc-1, Capan-1, and Capan-2 have been used in all experiments. S1R and TMEM97/S2R expression in PC cell lines was quantified by Real-Time qRT-PCR and Western Blot experiments. MTS assay was used to assess the antiproliferative effect of RC-106. The apoptotic properties of RC-106 was evaluated by TUNEL and caspase activation assays. GRP78/BiP, ATF4, and CHOP was quantified to evaluate ER-UPR. Proteasome activity was investigated by a specific fluorescent-based assay. Scratch wound healing assay was used to asses RC-106 effect on cell migration. In addition, we delineated the in vivo pharmacokinetic profile and pancreas distribution of RC-106 in male CD-1 mice. Results: Panc-1, Capan-1, and Capan-2 express both SRs. RC-106 exerts an antiproliferative and pro-apoptotic effect in all examined cell lines. Cells exposure to RC-106 induces the increase of the expression of ER-UPR related proteins, and the inhibition of proteasome activity. Moreover, RC-106 is able to decrease PC cell lines motility. The in vivo results show that RC-106 is more concentrated in pancreas than plasma. Conclusion: Overall, our data evidenced that the pan-SR modulator RC-106 is an optimal candidate for in vivo studies in animal models of PC

Genetic and epigenetic alterations of cdh1 regulatory regions in hereditary and sporadic gastric cancer

E-cadherin is a key player in gastric cancer (GC) and germline alterations of CDH1, its encoding gene, are responsible for Hereditary Diffuse Gastric Cancer (HDGC) syndrome. This study aimed at elucidating the role of genetic variants and DNA methylation of CDH1 promoter and enhancers in the regulation of gene expression. For this purpose, we analyzed genetic variants of the CDH1 gene through Next-Generation Sequencing (NGS) in a series of GC cell lines (NCI-N87, KATO-III, SNU-1, SNU-5, GK2, AKG, KKP) and the corresponding CDH1 expression levels. By bisulfite genomic sequencing, we analyzed the methylation status of CDH1 regulatory regions in 8 GC cell lines, in a series of 13 sporadic GC tissues and in a group of 20 HDGC CDH1-negative patients and 6 healthy controls. The NGS analysis on CDH1 coding and regulatory regions detected genetic alterations in 3 out of 5 GC cell lines lacking functional E-cadherin. CDH1 regulatory regions showed different methylation patterns in patients and controls, GC cell lines and GC tissues, expressing different E-cadherin levels. Our results showed that alterations in terms of genetic variants and DNA methylation patterns of both promoter and enhancers are associated with CDH1 expression levels and have a role in its regulation.This research and its authors were funded by IRCCS IRST (G.T., C.M., R.D. V.A., M.R., F.R., M.C., S.P., G.M., D.C., P.U.) and by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020–Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT–Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) (C.S.J., R.B.-M., A.A., C.O.). This work was also financed by the project NORTE-01-0145-FEDER-000029 (CANCER)-supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)–project POCI-01-0145-FEDER-016390 (CancelStem) and PTDC/BTM-TEC/30164/2017 (3DChroMe), funded by ERDF, POCI and FCT

Examining the benefits and barriers for the implementation of Net Zero Energy settlements

The transition of the Net Zero Energy (NZE) concept from building to settlement scale has been theoretically approached in a number of studies. This paper examines the benefits and barriers associated with the implementation of the NZE concept at a settlement scale, by adopting a comprehensive approach for the design, construction, and monitoring of NZE settlements that was developed in the EU Horizon 2020 ZERO-PLUS project and implemented in four case studies. First, the ZERO-PLUS approach is presented, followed by an analysis of associated benefits and encountered barriers. Next, the roles of different stakeholders involved in the process are identified through stakeholder analysis. Finally, new dynamics that emerge and are critical to the successful implementation of NZE settlements are discussed. The ZERO-PLUS approach leads to achieving NZE settlements with an initial cost that is on average 16% lower than the cost of a typical NZEB, while achieving a net regulated energy consumption of less than 20 kWh/m2/year and renewable energy production of more than 50 kWh/m2/year. The implementation of NZE settlements revealed two main issues: 1) the external barriers that were raised by the planning policies and regulations; and 2) the challenge of managing and integrating the needs and requirements of project stakeholders. To overcome these barriers while reaping the benefits of the approach, the management of such projects needs to focus from the outset on the establishment of a project management structure that will ensure the coordination and integration of various stakeholders. The use of a standardized collaboration protocol from the preliminary design stage is recommended to facilitate future projects. Simultaneously, regulations need to be updated towards facilitating NZE settlement implementation

Pegvisomant in acromegaly: a multicenter real-life study in Argentina

OBJECTIVE: To describe the long term safety and efficacy of pegvisomant (PEGV), and the predictors of treatment response in patients with acromegaly in the real life setting. SUBJECTS AND METHODS: We retrospectively reviewed the clinical, hormonal and radiological data of acromegalic patients treated with PEGV in 17 Argentine centers. RESULTS: Seventy-five patients (age range 22-77, 51 females) with acromegaly have been treated with PEGV for up to 118 months (median 27 months). Before PEGV, 97.3% of patients had been treated with medical therapy, surgery and/or radiotherapy, two patients had no previous treatment. At that time, all patients had an IGF-1 above the upper normal limit (ULN) (mean 2.4 x ULN ± 0.98, range 1.25-7). At diagnosis of acromegaly 84% presented macroadenomas, prior to PEGV only 23,5% of patients remained with tumor remnant > 1 cm, the remaining showed normal or less than 1 cm images. Disease control (IGF-1 ≤ 1.2 x ULN) was achieved in 62.9% of patients with a mean dose of 11.8 mg/day. Thirty-four patients (45%) received PEGV monotherapy, while 41 (55%) received combined therapy with either somatostatin analogues and/or cabergoline. Adverse events related to PEGV were: local injection site reaction in 5.3%, elevated liver enzymes in 9.3%, and tumor size growth in 9.8%. Pre-PEGV IGF-I level was the only predictor of treatment response: 2.1 x ULN vs 2.8 x ULN in controlled and uncontrolled patients respectively (p < 0.001). CONCLUSION: this long term experience indicates PEGV treatment was highly effective and safe in our series of Argentine patients with acromegaly refractory to standard therapies. Arch Endocrinol Metab. 2019;63(4):320-7.FIl: Garcia Basavilbaso, Natalia Ximena. Hospital Carlos G. Durand. Departamento de Endocrinología; Argentina.FIl: Ballarino, Maria Carolina. Hospital Militar Central. Servicio de Endocrinología; Argentina.FIl: Bruera, Darío. Clínica Caraffa. Servicio de Endocrinología; Argentina.FIl: Bruno, Oscar D. Hospital de Clínicas "José de San Martín". Servicio de Endocrinología; Argentina.FIl: Chervin, Alberto B. Hospital Santa Lucía. Servicio de Endocrinología; Argentina.FIl: Danilowicz, Karina. Hospital de Clínicas José de San Martín. Servicio de Endocrinología; Argentina.FIl: Fainstein Day, Patricia. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Fidalgo, Silvina Gabriela. Hospital Churruca. Servicio de Endocrinología; Argentina.FIl: Frigeri, Adriana. Hospital Teodoro Alvarez. Servicio de Endocrinología; Argentina.FIl: Glerean, Mariela. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Guelman, Rodolfo. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Isaac, Gabriel. Hospital Privado de la Comunidad. Servicio de Endocrinología; Argentina.FIl: Knoblovits, Pablo. Hospital Italiano. Servicio de Endocrinología; Argentina.FIl: Librandi, Fabiana. Hospital Rivadavia. Servicio de Endocrinología; Argentina.FIl: López Montes, Monica. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas “José de San Martín”. Servicio de Endocrinología; Argentina.FIl: Mallea-Gil, María Susana. Hospital Militar Central. Servicio de Endocrinología; Argentina.FIl: Manavela, Marcos. Hospital de Clínicas “José de San Martín”. Servicio de Endocrinología; Argentina.FIl: Mereshian, Paula. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas “José de San Martín”. Servicio de Endocrinología; Argentina.FIl: Moncet, Daniel. Hospital Privado de la Comunidad. Servicio de Endocrinología; Argentina.FIl: Katz, Debora Adela. Fleni. Departamento de Neurología. Servicio de Neuroendocrinología; Argentina.FIl: Pignatta, Analia. Hospital Interzonal San Juan Bautista. Servicio de Endocrinología; Argentina.FIl: Rogozinsky, Amelia. Hospital Ramos Mejía. Servicio de Endocrinología; Argentina.FIl: Sago, Laura R. Hospital Italiano de La Plata. Servicio de Endocrinología; Argentina.FIl: Servidio, Marisa. Hospital Teodoro Alvarez. Servicio de Endocrinología; Argentina.FIl: Spezzi, Monica. Instituto Médico Platense. Servicio de Endocrinología; Argentina.FIl: Stalldecker, Graciela. Hospital Pirovano; Argentina.FIl: Tkatch, Julieta. Hospital Carlos G. Durand. Departamento de Endocrinología; Argentina.FIl: Vitale, Nicolas Marcelo. Hospital Santa Lucía. Servicio de Endocrinología; Argentina.FIl: Guitelman, Mirtha. Hospital Carlos G. Durand. Departamento de Endocrinología; Argentina

Second-Line Medications for Women Aged 10 to 50 Years with Idiopathic Generalized Epilepsy

Importance: Women with idiopathic generalized epilepsy (IGE) face challenges in treatment due to limited options that are both effective and safe. Objective: To evaluate the effectiveness and safety of substitution monotherapy vs add-on therapy as second-line options for women who might become pregnant with IGE after failure of first-line antiseizure medications (ASMs) other than valproic acid. Design, Setting, and Participants: Multicenter retrospective comparative effectiveness cohort study at 18 primary, secondary, and tertiary adult and children epilepsy centers across 4 countries, analyzing data from 1995 to 2023. Participants were women aged 10 to 50 years diagnosed with IGE who were prescribed a second line of ASM. Main Outcomes and Measures: Treatment failure (TF), defined as the replacement or addition of a second ASM due to ineffectiveness, was compared between patients receiving ASM add-on or substitution monotherapy using inverse probability of treatment weighting (IPTW)-adjusted Cox proportional hazards regression. Exploratory analyses were also conducted to assess the effectiveness of individual ASMs and various ASM combinations. Results: This study included 249 women with a median (IQR) age of 18.0 (15.5-22.0) years. Among them, 146 (58.6%) received an add-on regimen, and 103 (41.4%) received substitution monotherapy. During follow-up, TF occurred in 48 patients (32.9%) receiving add-on therapy and 36 (35.0%) using substitution monotherapy, with no significant differences between groups (IPTW-adjusted hazard ratio [HR], 0.89; 95% CI, 0.53-1.51; P =.69). ASM discontinuation due to ineffectiveness or adverse effects occurred in 36 patients (24.7%) receiving add-on therapy and 29 (28.2%) receiving substitution monotherapy, showing no significant differences (IPTW-adjusted HR, 0.97; 95% CI, 0.57-1.65; P =.92). Rates of ASM discontinuation due to adverse effects only were low in both groups, occurring in 13 patients (9.0%) receiving add-on therapy and 9 (8.7%) receiving a substitution monotherapy. Among add-on regimens other than valproic acid, the combination of levetiracetam and lamotrigine demonstrated a lower risk of TF compared with other combinations with levetiracetam plus other ASM (adjusted HR, 2.41; 95% CI, 1.12-5.17; P =.02) and lamotrigine plus other ASM (adjusted HR, 4.03; 95% CI, 1.73-9.39; P =.001). However, valproic acid remained the most effective second-line ASM when considering individual agents. Conclusions and relevance: In this comparative effectiveness study of second-line treatment strategies for women with IGE, no significant differences were observed between substitution monotherapy and add-on therapy

Effectiveness and safety of adjunctive cenobamate in people with focal-onset epilepsy: Interim results after 24-week observational period from the BLESS study

Objective: Cenobamate is an antiseizure medication (ASM) with a dual mechanism of action that was recently approved for the treatment of focal seizures in adults. This analysis aimed to describe the outcomes at 12 and 24 weeks after starting cenobamate therapy in a real-world setting. Methods: BLESS [NCT05859854] is an ongoing, observational, retrospective and prospective cohort study to evaluate the real-world effectiveness and safety of adjunctive cenobamate in adults with uncontrolled focal epilepsy. Subgroup analysis was performed in subjects with 2 to 3 previous ASMs (early users) and those with &gt;3 previous ASMs (late users). Results: The second interim analysis of the BLESS study included 388 participants with a median (interquartile range) age of 43.0 (31.0-54.0) years. They had a median of 6.0 (4.0-9.0) prior ASMs and a median of 7.2 (3.0-20.6) monthly seizures at baseline. The median monthly seizure frequency was reduced by 59.9% (19.2%-87.3%) from baseline to 24 weeks; 229 (59.0%) subjects had a &gt;= 50% seizure frequency reduction, and 44 (11.3%) showed sustained seizure freedom. The proportion of participants taking &lt;= 2 concomitant ASMs increased from 217 (56.5%) at baseline to 239 (65.7%) at 24 weeks. Among the early users (n = 76, 19.6%), the median reduction in monthly seizure frequency at 24 weeks was 78.0% (50.0-97.1%), and 76.3% of subjects had a &gt;= 50% response rate. The frequency of adverse drug reactions (ADRs) was 5.3% and 23.4% in early and late users. The most frequent ADRs were somnolence, dizziness, and balance disorder; after the occurrence of ADRs, 63.5% of participants maintained the prescribed dose, and 5.2% permanently discontinued treatment. Significance: Cenobamate was effective in reducing seizure frequency in a real-world setting and showed a manageable safety profile. The treatment with cenobamate also reduced the burden of concomitant ASMs in both early and late users

Adjunctive Brivaracetam in People with Epilepsy and Intellectual Disability: Evidence from the BRIVAracetam Add-On First Italian netwoRk Study

Introduction: Subjects with intellectual disability are usually excluded from clinical trials and there is limited evidence-based guidance for the choice of antiseizure medications in this vulnerable population. The study explored the effectiveness of brivaracetam (BRV) in people with epilepsy and intellectual disability. Methods: BRIVAracetam add-on First Italian netwoRk Study (BRIVAFIRST) was a 12-month retrospective, multicenter study including adults prescribed adjunctive BRV. Main outcomes included the rates of seizure‐freedom, seizure response (≥&nbsp;50% reduction in baseline seizure frequency), and treatment discontinuation. The occurrence of adverse events (AEs) was also considered. Analyses by the presence and severity of intellectual disability were performed. Results: Subjects with intellectual disability were 253 (24.6%) out of 1029 participants. The 12-month rates of seizure freedom were 18.4% and 10.3% in participants without and with intellectual disability, respectively; the corresponding values for seizure response were 40.0% and 28.9%. Intellectual disability was not an independent predictor of seizure outcomes. The rates of treatment discontinuation were 25.8% and 26.4% in participants without and with intellectual disability. respectively. There were no statistically significant differences in the rates of any AEs, somnolence, nervousness/agitation, and aggressiveness by the presence and degree of intellectual disability. Conclusion: Brivaracetam can be a suitable treatment option and offer opportunities for clinical improvement in subjects with intellectual disability and uncontrolled seizures

Adjunctive Brivaracetam in Focal Epilepsy: Real-World Evidence from the BRIVAracetam add-on First Italian netwoRk STudy (BRIVAFIRST)

Background: In randomized controlled trials, add-on brivaracetam (BRV) reduced seizure frequency in patients with drug-resistant focal epilepsy. Studies performed in a naturalistic setting are a useful complement to characterize the drug profile. Objective: This multicentre study assessed the effectiveness and tolerability of adjunctive BRV in a large population of patients with focal epilepsy in the context of real-world clinical practice. Methods: The BRIVAFIRST (BRIVAracetam add-on First Italian netwoRk STudy) was a retrospective, multicentre study including adult patients prescribed adjunctive BRV. Patients with focal epilepsy and 12-month follow-up were considered. Main outcomes included the rates of seizure‐freedom, seizure response (≥&nbsp;50% reduction in baseline seizure frequency), and treatment discontinuation. The incidence of adverse events (AEs) was also considered. Analyses by levetiracetam (LEV) status and concomitant use of strong enzyme-inducing antiseizure medications (EiASMs) and sodium channel blockers (SCBs) were performed. Results: A total of 1029 patients with a median age of 45&nbsp;years (33–56) was included. At 12 months, 169 (16.4%) patients were seizure-free and 383 (37.2%) were seizure responders. The rate of seizure freedom was 22.3% in LEV-naive patients, 7.1% in patients with prior LEV use and discontinuation due to insufficient efficacy, and 31.2% in patients with prior LEV use and discontinuation due to AEs (p&nbsp;&lt;&nbsp;0.001); the corresponding values for ≥&nbsp;50% seizure frequency reduction were 47.9%, 29.7%, and 42.8% (p&nbsp;&lt;&nbsp;0.001). There were no statistically significant differences in seizure freedom and seizure response rates by use of strong EiASMs. The rates of seizure freedom (20.0% vs. 16.6%; p&nbsp;=&nbsp;0.341) and seizure response (39.7% vs. 26.9%; p&nbsp;=&nbsp;0.006) were higher in patients receiving SCBs than those not receiving SCBs; 265 (25.8%) patients discontinued BRV. AEs were reported by 30.1% of patients, and were less common in patients treated with BRV and concomitant SCBs than those not treated with SCBs (28.9% vs. 39.8%; p&nbsp;=&nbsp;0.017). Conclusion: The BRIVAFIRST provided real-world evidence on the effectiveness of BRV in patients with focal epilepsy irrespective of LEV history and concomitant ASMs, and suggested favourable therapeutic combinations

Sustained seizure freedom with adjunctive brivaracetam in patients with focal onset seizures

The maintenance of seizure control over time is a clinical priority in patients with epilepsy. The aim of this study was to assess the sustained seizure frequency reduction with adjunctive brivaracetam (BRV) in real-world practice. Patients with focal epilepsy prescribed add-on BRV were identified. Study outcomes included sustained seizure freedom and sustained seizure response, defined as a 100% and a ≥50% reduction in baseline seizure frequency that continued without interruption and without BRV withdrawal through the 12-month follow-up. Nine hundred ninety-four patients with a median age of 45 (interquartile range = 32–56) years were included. During the 1-year study period, sustained seizure freedom was achieved by 142 (14.3%) patients, of whom 72 (50.7%) were seizure-free from Day 1 of BRV treatment. Sustained seizure freedom was maintained for ≥6, ≥9, and 12&nbsp;months by 14.3%, 11.9%, and 7.2% of patients from the study cohort. Sustained seizure response was reached by 383 (38.5%) patients; 236 of 383 (61.6%) achieved sustained ≥50% reduction in seizure frequency by Day 1, 94 of 383 (24.5%) by Month 4, and 53 of 383 (13.8%) by Month 7 up to Month 12. Adjunctive BRV was associated with sustained seizure frequency reduction from the first day of treatment in a subset of patients with uncontrolled focal epilepsy