Competition between Self-Assembly and Phase Separation Governs High-Temperature Condensation of a DNA Liquid.

In many biopolymer solutions, attractive interactions that stabilize finite-sized clusters at low concentrations also promote phase separation at high concentrations. Here we study a model biopolymer system that exhibits the opposite behavior, whereby self-assembly of DNA oligonucleotides into finite-sized, stoichiometric clusters tends to inhibit phase separation. We first use microfluidics-based experiments to map a novel phase transition in which the oligonucleotides condense as the temperature increases at high concentrations of divalent cations. We then show that a theoretical model of competition between self-assembly and phase separation quantitatively predicts changes in experimental phase diagrams arising from DNA sequence perturbations. Our results point to a general mechanism by which self-assembly shapes phase boundaries in complex biopolymer solutions.

Cell-mediated cytotoxicity within CSF and brain parenchyma in spinal muscular atrophy unaltered by nusinersen treatment.

5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.

POSoligo software for in vitro gene synthesis.

Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.

Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection.

Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B Streptococcus (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms.

Synthesis and Properties of Oligonucleotides Containing LNA-Sulfamate and Sulfamide Backbone Linkages.

Oligonucleotides hold great promise as therapeutic agents but poor bioavailability limits their utility. Hence, new analogues with improved cell uptake are urgently needed. Here, we report the synthesis and physical study of reduced-charge oligonucleotides containing artificial LNA-sulfamate and sulfamide linkages combined with 2'-O-methyl sugars and phosphorothioate backbones. These oligonucleotides have high affinity for RNA and excellent nuclease resistance.

Profiling tofersen as a treatment of superoxide dismutase 1 amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive motor neuron disorder with a fatal outcome 3-5 years after disease onset due to respiratory complications. Superoxide dismutase 1 (SOD1) mutations are found in about 2% of all patients. Tofersen is a novel oligonucleotide antisense drug specifically developed to treat SOD1-ALS patients. AREAS COVERED: Our review covers and discusses tofersen pharmacological properties and its phase I/II and III clinical trials results. Other available drugs and their limitations are also addressed. EXPERT OPINION: VALOR study failed to meet the primary endpoint (change in the revised Amyotrophic Lateral Sclerosis Functional Rating Scale score from baseline to week 28, tofersen arm vs. placebo), but a significant reduction in plasma neurofilament light chain (NfL) levels was observed in tofersen arm (60% vs. 20%). PrefALS study has proposed plasma NfL has a potential biomarker for presymptomatic treatment, since it increases 6-12 months before phenoconversion. There is probably a delay between plasma NfL reduction and the clinical benefit. ATLAS study will allow more insights regarding tofersen clinical efficacy in disease progression rate, survival, and even disease onset delay in presymptomatic SOD1 carriers.

Sequence confirmation of synthetic DNA exceeding 100 nucleotides using restriction enzyme mediated digestion combined with high-resolution tandem mass spectrometry.

Oligonucleotides have emerged as important therapeutic options for inherited diseases. In recent years, RNA therapeutics, especially mRNA, have been pushed to the market. Analytical methods for these molecules have been published extensively in the last few years. Notably, mass spectrometry has proven as a state-of-the-art quality control method. For RNA based therapeutics, numerous methods are available, while DNA therapeutics lack of suitable MS-based methods when it comes to molecules exceeding approximately 60 nucleotides. We present a method which combines the use of common restriction enzymes and short enzyme-directing oligonucleotides to generate DNA digestion products with the advantages of high-resolution tandem mass spectrometry. The instrumentation includes ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer with a collision induced dissociation (CID) for sequence analysis. Utilizing this approach, we increased the sequence coverage from 23.3% for a direct CID-MS/MS experiment of a 100 nucleotide DNA molecule to 100% sequence coverage using the restriction enzyme mediated approach presented in this work. This approach is suitable for research and development and quality control purposes in a regulated environment, which makes it a versatile tool for drug development.

Improvement of oligonucleotide separation using a repetto high-performance liquid chromatography recycling approach.

A new approach for the improvement of separation of oligonucleotides by recycling ion-pairing chromatography is described. In the so-called repetto process, segments of separated compounds are sequentially returned to the inlet for multiple passages through the column without a need to pass a pump and with the possibility of detecting the level of separation between individual passages. Unlike in the recently described twin-column recycle approach in which eluents are repeatedly transferred between two separation columns, with the repetto method a single column is sufficient, and the detector is not exposed to high back pressure. The repetto principle was used for the separation of synthetic oligonucleotides, resulting in a multi-fold improvement in single nt resolution of long (> 50 nt) synthetic oligonucleotide fragments with high gas chromatography (guanine-cytosine) content > 40% and their separation from impurities of the original synthesis.

Long-Term Comparative Efficacy and Safety of Risdiplam and Nusinersen in Children with Type 1 Spinal Muscular Atrophy.

INTRODUCTION: Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disease characterized by a loss of motor neurons and progressive muscle weakness. Children with untreated type 1 SMA never sit independently and require increasing levels of ventilatory support as the disease progresses. Without intervention, and lacking ventilatory support, death typically occurs before the age of 2 years. There are currently no head-to-head trials comparing available treatments in SMA. Indirect treatment comparisons are therefore needed to provide information on the relative efficacy and safety of SMA treatments for healthcare decision-making. METHODS: The long-term efficacy and safety of risdiplam versus nusinersen in children with type 1 SMA was evaluated using indirect treatment comparison methodology to adjust for differences between population baseline characteristics, to reduce any potential bias in the comparative analysis. An unanchored matching-adjusted indirect comparison was conducted using risdiplam data from 58 children in FIREFISH (NCT02913482) and published aggregate nusinersen data from 81 children obtained from the ENDEAR (NCT02193074) and SHINE (NCT02594124) clinical trials with at least 36 months of follow-up. RESULTS: Children with type 1 SMA treated with risdiplam had a 78% reduction in the rate of death, an 81% reduction in the rate of death or permanent ventilation, and a 57% reduction in the rate of serious adverse events compared with children treated with nusinersen. Children treated with risdiplam also had a 45% higher rate of achieving a Hammersmith Infant Neurological Examination, Module 2 motor milestone response and a 186% higher rate of achieving a ≥ 4-point improvement in Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders compared with children treated with nusinersen. CONCLUSION: Long-term data supported risdiplam as a superior alternative to nusinersen in children with type 1 SMA. Video abstract available for this article. Video abstract (MP4 184542 KB).

Risdiplam and nusinersen are two approved treatments for patients with type 1 spinal muscular atrophy (SMA). There are currently no head-to-head trials that compare the outcomes of these treatments in patients. This study conducted a statistical comparison of the efficacy and safety of risdiplam and nusinersen in children with type 1 SMA who received treatment for at least 36 months. Risdiplam data were collected from 58 children who participated in the FIREFISH trial (NCT02913482). Published combined data were collected from 81 children treated with nusinersen who participated in the ENDEAR (NCT02193074) and SHINE (NCT02594124) trials. Outcomes from the two studies were compared using matching-adjusted indirect comparison (MAIC) methodology. MAIC adjusts for differences in baseline characteristics between patients in two trials to make the populations more similar and reduce bias in the comparison. Results suggested that children with type 1 SMA treated with risdiplam had a 78% reduction in the rate of death and an 81% reduction in the rate of death or permanent ventilation compared with children treated with nusinersen. With risdiplam, children also had a higher rate of achieving motor function responses, and a longer time to the first serious adverse event compared with children treated with nusinersen. These results support risdiplam as a superior alternative to nusinersen in children with type 1 SMA over 36 months of follow-up. Access to long-term data beyond 36 months would allow for additional indirect comparisons between SMA treatments. These comparisons are key to guiding treatment decision-making in the absence of head-to-head trials.

In vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis.

Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With the ever-expanding application of directed evolution, different approaches of generating genetic diversity are required to enrich the traditional toolbox. Here we show in vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis (epADS). This approach comprises a three-step process which incorporates base errors randomly generated during chemical synthesis of oligonucleotides under specific conditions into the target DNA. Through this method, 200 ~ 4000 folds of diversification in fluorescent strength have been achieved in genes encoding fluorescent proteins. EpADS has also been successfully used to diversify regulatory genetic parts, synthetic gene circuits and even increase microbial tolerance to carbenicillin in a short time period. EpADS would be an alternative tool for directed evolution which may have useful applications in synthetic biology.

A sensitive analytical strategy of oligonucleotide functionalized fluorescent probes for detection of nusinersen sodium in human serum.

Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disease. Nusinersen sodium (NS) is the world's first antisense oligonucleotide (ASO) drug for SMA precise targeted therapy. However, the limited half-life of oligonucleotides and their tendency to accumulate in hepatic and renal tissues presented significant challenges for clinical investigation and therapeutic drug monitoring. In this study, we proposed an analytical strategy based on the specific capture of oligonucleotide functionalized fluorescent probes by single stranded binding proteins (SSB) for ultra-sensitive and high-throughput detection of nusinersen sodium in human serum. The magnetic nanoparticles modified with single-strand binding protein (MNPs-SSB) selectively bonded to the red fluorescent quantum dots functionalized with oligonucleotides (RQDs-ssDNA) that were complementary to nusinersen sodium. Upon interaction with nusinersen sodium, RQDs-ssDNA formed a double-stranded complex (RQDs-ssDNA-NS), resulting in enhanced red fluorescence after magnetic separation as it was no longer captured by MNPs-SSB but remained in the supernatant. A quantitative analysis of nusinersen sodium in biological samples was successfully achieved by establishing a relationship between fluorescence intensity and its concentration. The detection signal F/F0 exhibited a linear correlation (R2 = 0.9871) over a wide range from 0.1 nM to 200 nM, with a limit of detection (LOD) of 0.03 nM, demonstrating the high specificity and rapid analysis time (only 30 min). This method provided a novel approach for sensitive, high-throughput, and specific analysis of nusinersen sodium and similar ASO drugs.

Modern approaches to therapeutic oligonucleotide manufacturing.

Therapeutic oligonucleotides are a powerful drug modality with the potential to treat many diseases. The rapidly growing number of therapies that have been approved and that are in advanced clinical trials will place unprecedented demands on our capacity to manufacture oligonucleotides at scale. Existing methods based on solid-phase phosphoramidite chemistry are limited by their scalability and sustainability, and new approaches are urgently needed to deliver the multiton quantities of oligonucleotides that are required for therapeutic applications. The chemistry community has risen to the challenge by rethinking strategies for oligonucleotide production. Advances in chemical synthesis, biocatalysis, and process engineering technologies are leading to increasingly efficient and selective routes to oligonucleotide sequences. We review these developments, along with remaining challenges and opportunities for innovations that will allow the sustainable manufacture of diverse oligonucleotide products.

IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution.

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Neurophysiological Characteristics in Type II and Type III 5q Spinal Muscular Atrophy Patients: Impact of Nusinersen Treatment.

Objective: This study aimed to observe the neurophysiological characteristics of type II and type III 5q spinal muscular atrophy (SMA) patients and the changes in peripheral motor nerve electrophysiology after Nusinersen treatment, as well as the influencing factors. Methods: This single-center retrospective case-control study collected clinical data and peripheral motor nerve CMAP parameters from 42 5qSMA patients and 42 healthy controls at the Second Affiliated Hospital of Xi'an Jiaotong University (January 2021 to December 2022). It evaluated changes in motor function and CMAP amplitude before and after Nusinersen treatment. Results: Our investigation encompassed all symptomatic and genetically confirmed SMA patients, consisting of 32 type II and 10 type III cases, with a median age of 57 months (29.5 to 96 months). Comparative analysis with healthy controls revealed substantial reductions in CMAP amplitudes across various nerves in both type II and type III patients. Despite the administration of Nusinersen treatment for 6 or 14 months to the entire cohort, discernible alterations in motor nerve amplitudes were not observed, except for a significant improvement in younger patients (≤36 months) at the 14-month mark. Further scrutiny within the type II subgroup unveiled that individuals with a disease duration ≤12 months experienced a noteworthy upswing in femoral nerve amplitude, a statistically significant difference when compared to those with >12 months of disease duration. Conclusion: Motor nerve amplitudes were significantly decreased in type II and type III 5q SMA patients compared to healthy controls. Nusinersen treatment showed better improvement in motor nerve amplitudes in younger age groups and those with shorter disease duration, indicating a treatment-time dependence.

Cross-Reactive Polyclonal Antibodies Raised Against GalNAc-Conjugated siRNA Recognize Mostly the GalNAc Moiety.

Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.

Real-life experience with inotersen at CEPARM, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro.

BACKGROUND: Hereditary transthyretin amyloidosis (ATTRv) is an inherited, progressive, and fatal disease still largely underdiagnosed. Mutations in the transthyretin (TTR) gene cause the TTR protein to destabilize, misfold, aggregate, and deposit in body tissues, which makes ATTRv a disease with heterogeneous clinical phenotype. OBJECTIVE: To describe the long-term efficacy and safety of inotersen therapy in patients with ATTRv peripheral neuropathy (ATTRv-PN). METHODS: Patients who completed the NEURO-TTR pivotal study and the NEURO-TTR OLE open-label extension study migrated to the present study and were followed-up for at least 18 more months to an average of 67 months and up to 76 months since day 1 of the inotersen therapy (D1-first dose of inotersen). Disease progression was evaluated by standard measures. RESULTS: Ten ATTRv-PN patients with Val30Met mutation were included. The mean disease duration on D1 was of 3 years, and the mean age of the patients was of 46.8 years. During an additional 18-month follow up, neurological function, based on the Neuropathy Impairment Score and the Polyneuropathy Disability Score, functionality aspects (Karnofsky Performance Status), and nutritional and cardiac aspects were maintained. No new safety signs have been noted. CONCLUSION: The treatment with inotersen was effective and well tolerated for the average of 67 months and up to 76 months. Our results are consistent with those of larger phase-III trials.

ANTECEDENTES: Amiloidose hereditária por transtirretina (ATTRv) é uma doença hereditária, progressiva e fatal ainda largamente subdiagnosticada. Mutações no gene transtirretina (TTR) promovem desestabilização, desdobramento, agregação e depósito da proteína TTR em tecidos do corpo, o que faz da ATTRv uma doença de fenótipo clínico heterogêneo. OBJETIVO: Descrever a eficácia e segurança da terapia com inotersena no longo prazo em pacientes com neuropatia periférica ATTRv (ATTRv-PN). MéTODOS: Pacientes que completaram o estudo pivotal NEURO-TTR e o estudo de extensão aberta NEURO-TTR OLE migraram para este estudo e foram acompanhados por no mínimo 18 meses adicionais, em média por 67 meses, e por até 76 meses, desde o dia 1 da terapia com inotersena (D1­primeira dose de inotersena). A progressão da doença foi avaliada por medidas padronizadas. RESULTADOS: Dez pacientes com ATTRv-PN com mutação Val30Met foram incluídos. A duração média da doença no D1 era de 3 anos, e a média de idade dos pacientes era de 46,8 anos. Durante o período de acompanhamento adicional de 18 meses, a função neurológica, baseada no Neuropathy Impairment Score e no Polyneuropathy Disability Score, os aspectos de funcionalidade (Karnofsky Performance Status), nutricional e cardíacos estavam mantidos. Não se observou nenhum novo sinal de segurança. CONCLUSãO: O tratamento com inotersena foi eficaz e bem tolerado por 67 meses em média, e por até 76 meses. Nossos resultados são consistentes com os de estudos maiores de fase III.

Mixed insect pest populations of Diaspididae species under control of oligonucleotide insecticides: 3'-end nucleotide matters.

Diaspididae are one of the most serious small herbivorous insects with piercing-sucking mouth parts and are major economic pests as they attack and destroy perennial ornamentals and food crops. Chemical control is the primary management approach for armored scale infestation. However, chemical insecticides do not possess selectivity in action and not always effective enough for the control of armored scale insects. Our previous work showed that green oligonucleotide insecticides (olinscides) are highly effective against armored and soft scale insects. Moreover, olinscides possess affordability, selectivity in action, fast biodegradability, and a low carbon footprint. Insect pest populations undergo microevolution and olinscides should take into account the problem of insecticide resistance. Using sequencing results, it was found that in the mixed populations of insect pests Dynaspidiotus britannicus Newstead and Aonidia lauri Bouche, predominates the population of A. lauri. Individuals of A. lauri comprised for 80% of individuals with the sequence 3'-ATC-GTT-GGC-AT-5' in the 28S rRNA site, and 20% of the population comprised D. britannicus individuals with the sequence 3'-ATC-GTC-GGT-AT-5'. We created olinscides Diasp80-11 (5'-ATG-CCA-ACG-AT-3') and Diasp20-11 (5'-ATA-CCG-ACG-AT-3') with perfect complementarity to each of the sequences. Mortality of insects on the 14th day comprised 98.19 ± 3.12% in Diasp80-11 group, 64.66 ± 0.67% in Diasp20-11 group (p < 0.05), and 3.77 ± 0.94% in the control group. Results indicate that for maximum insecticidal effect it is necessary to use an oligonucleotide insecticide that corresponds to the dominant species. Mortality in Diasp80-11 group was accompanied with significant decrease in target 28S rRNA concentration and was 8.44 ± 0.14 and 1.72 ± 0.36 times lower in comparison with control (p < 0.05) on the 10th and 14th days, respectively. We decided to make single nucleotide substitutions in Diasp20-11 olinscide to understand which nucleotide will play the most important role in insecticidal effect. We created three sequences with single nucleotide transversion substitutions at the 5'-end - Diasp20(5')-11 (A to T), 3'-end - Diasp20(3')-11 (T to A), and in the middle of the sequence - Diasp20(6)-11 (6th nitrogenous base of the sequence; G to C), respectively. As a result, mortality of mixed population of the field experiment decreased and comprised 53.89 ± 7.25% in Diasp20(5')-11 group, 40.68 ± 4.33% in Diasp20(6)-11 group, 35.74 ± 5.51% in Diasp20(3')-11 group, and 3.77 ± 0.94% in the control group on the 14th day. Thus, complementarity of the 3'-end nucleotide to target 28S rRNA was the most important for pronounced insecticidal effect (significance of complementarity of nucleotides for insecticidal effect: 5' nt < 6 nt < 3' nt). As was found in our previous research works, the most important rule to obtain maximum insecticidal effect is complete complementarity to the target rRNA sequence and maximum coverage of target sequence in insect pest populations. However, in this article we also show that the complementarity of 3'-end is a second important factor for insecticidal potential of olinscides. Also in this article we propose 2-step DNA containment mechanism of action of olinscides, recruiting RNase H. The data obtained indicate the selectivity of olinscides and at the same time provide a simple and flexible platform for the creation of effective plant protection products, based on antisense DNA oligonucleotides.

MicroRNA Expression Profiles in Human Samples and Cell Lines Revealed Nine miRNAs Associated with Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

Metastasis and drug resistance are major contributors to cancer-related fatalities worldwide. In ovarian cancer (OC), a staggering 70% develop resistance to the front-line therapy, cisplatin. Despite proposed mechanisms, the molecular events driving cisplatin resistance remain unclear. Dysregulated microRNAs (miRNAs) play a role in OC initiation, progression, and chemoresistance, yet few studies have compared miRNA expression in OC samples and cell lines. This study aimed to identify key miRNAs involved in the cisplatin resistance of high-grade-serous-ovarian-cancer (HGSOC), the most common gynecological malignancy. MiRNA expression profiles were conducted on RNA isolated from formalin-fixed-paraffin-embedded human ovarian tumor samples and HGSOC cell lines. Nine miRNAs were identified in both sample types. Targeting these with oligonucleotide miRNA inhibitors (OMIs) reduced proliferation by more than 50% for miR-203a, miR-96-5p, miR-10a-5p, miR-141-3p, miR-200c-3p, miR-182-5p, miR-183-5p, and miR-1206. OMIs significantly reduced migration for miR-183-5p, miR-203a, miR-296-5p, and miR-1206. Molecular pathway analysis revealed that the nine miRNAs regulate pathways associated with proliferation, invasion, and chemoresistance through PTEN, ZEB1, FOXO1, and SNAI2. High expression of miR-1206, miR-10a-5p, miR-141-3p, and miR-96-5p correlated with poor prognosis in OC patients according to the KM plotter database. These nine miRNAs could be used as targets for therapy and as markers of cisplatin response.

Neurodegeneration Biomarkers in Adult Spinal Muscular Atrophy (SMA) Patients Treated with Nusinersen.

The objective of this study is to evaluate biomarkers for neurodegenerative disorders in adult SMA patients and their potential for monitoring the response to nusinersen. Biomarkers for neurodegenerative disorders were assessed in plasma and CSF samples obtained from a total of 30 healthy older adult controls and 31 patients with adult SMA type 2 and 3. The samples were collected before and during nusinersen treatment at various time points, approximately at 2, 6, 10, and 22 months. Using ELISA technology, the levels of total tau, pNF-H, NF-L, sAPPß, Aß40, Aß42, and YKL-40 were evaluated in CSF samples. Additionally, plasma samples were used to measure NF-L and total tau levels using SIMOA technology. SMA patients showed improvements in clinical outcomes after nusinersen treatment, which were statistically significant only in walkers, in RULM (p = 0.04) and HFMSE (p = 0.05) at 24 months. A reduction in sAPPß levels was found after nusinersen treatment, but these levels did not correlate with clinical outcomes. Other neurodegeneration biomarkers (NF-L, pNF-H, total tau, YKL-40, Aß40, and Aß42) were not found consistently changed with nusinersen treatment. The slow progression rate and mild treatment response of adult SMA types 2 and 3 may not lead to detectable changes in common markers of axonal degradation, inflammation, or neurodegeneration, since it does not involve large pools of damaged neurons as observed in pediatric forms. However, changes in biomarkers associated with the APP processing pathway might be linked to treatment administration. Further studies are warranted to better understand these findings.