Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS.

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.

In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.

ZIF-67-Derived Co/N-Doped Carbon-Functionalized MXene for Enhanced Electrochemical Sensing of Carbendazim.

Herein, ZIF-67-derived Co and N-doped carbon (Co/NC) particle-modified multilayer MXene (MXene@Co/NC) was developed as remarkable electrode material for carbendazim (CBZ) detection. MXene as a substrate provides an excellent conductive framework and plentiful accessibility sites. Co/NC particles embedding in MXene can not only prevent the interlayer stacking of MXene but also contribute a great deal of metal catalytic active sites and finally improve the adsorption and catalytic properties of the composite. Accordingly, the MXene@Co/NC electrode displays excellent electrocatalytic activity toward CBZ oxidation. Experimental parameters such as pH value, accumulation time, MXene@Co/NC modification volume and constituent materials' mass ratios were optimized. Under optimal conditions, the as-prepared sensor based on MXene@Co/NC holds a broad linearity range from 0.01 µM to 45.0 µM with a low limit of detection (LOD) of 3.3 nM (S/N = 3, S means the detection signal, while N represents the noise of the instrument). Moreover, the proposed sensor displays excellent anti-interference ability, superior reproducibility, excellent stability, and successfully achieves actual applications for CBZ detection in a lettuce sample.

Development of a sensitive trial-ready poly(GP) CSF biomarker assay for C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis.

OBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.

RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia.

The finding that a GGGGCC (G4C2) hexanucleotide repeat expansion in the chromosome 9 ORF 72 (C9ORF72) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) links ALS/FTD to a large group of unstable microsatellite diseases. Previously, we showed that microsatellite expansion mutations can be bidirectionally transcribed and that these mutations express unexpected proteins by a unique mechanism, repeat-associated non-ATG (RAN) translation. In this study, we show that C9ORF72 antisense transcripts are elevated in the brains of C9ORF72 expansion-positive [C9(+)] patients, and antisense GGCCCC (G2C4) repeat-expansion RNAs accumulate in nuclear foci in brain. Additionally, sense and antisense foci accumulate in blood and are potential biomarkers of the disease. Furthermore, we show that RAN translation occurs from both sense and antisense expansion transcripts, resulting in the expression of six RAN proteins (antisense: Pro-Arg, Pro-Ala, Gly-Pro; and sense: Gly-Ala, Gly-Arg, Gly-Pro). These proteins accumulate in cytoplasmic aggregates in affected brain regions, including the frontal and motor cortex, hippocampus, and spinal cord neurons, with some brain regions showing dramatic RAN protein accumulation and clustering. The finding that unique antisense G2C4 RNA foci and three unique antisense RAN proteins accumulate in patient tissues indicates that bidirectional transcription of expanded alleles is a fundamental pathologic feature of C9ORF72 ALS/FTD. Additionally, these findings suggest the need to test therapeutic strategies that target both sense and antisense RNAs and RAN proteins in C9ORF72 ALS/FTD, and to more broadly consider the role of antisense expression and RAN translation across microsatellite expansion diseases.

Comparison and evaluation of L. reuteri and L. rhamnosus-fermented egg yolk on the physicochemical and flavor properties of cookies.

The study aims to explore an effective approach to improve the sensory quality and consumer satisfaction of cookies in the food industry. L. reuteri and L. rhamnosus were chosen to ferment egg yolk and their effects on dough properties and physicochemical properties, flavor, texture, color, and sensory acceptability of cookies were studied. Results show that the utilization of fermented egg yolk significantly decreased baking loss and increased spread factor of cookies. GC-MS analysis indicates different Lactobacillus species enhanced cookie flavor through unique mechanisms. Texture analysis shows cookies prepared with L. rhamnosus-fermented egg yolk had significantly lower hardness (1807.12 g) than control cookies (2028.34 g). Sensory evaluation reveals the L. reuteri-fermented egg yolk significantly improved the overall acceptability of cookies by enhancing appearance, flavor, and mouthfeel scores. These findings have practical implications for food manufacturers seeking to enhance their product's quality and appeal, thereby gaining a competitive edge in the market.

Modulation of histone deacetylase 6 (HDAC6) nuclear import and tubulin deacetylase activity through acetylation.

The reversible acetylation of histones and non-histone proteins by histone acetyltransferases and deacetylases (HDACs) plays a critical role in many cellular processes in eukaryotic cells. HDAC6 is a unique histone deacetylase with two deacetylase domains and a C-terminal zinc finger domain. HDAC6 resides mainly in the cytoplasm and regulates many important biological processes, including cell migration and degradation of misfold proteins. HDAC6 has also been shown to localize in the nucleus to regulate transcription. However, how HDAC6 shuttles between the nucleus and cytoplasm is largely unknown. In addition, it is not clear how HDAC6 enzymatic activity is modulated. Here, we show that HDAC6 can be acetylated by p300 on five clusters of lysine residues. One cluster (site B) of acetylated lysine is in the N-terminal nuclear localization signal region. These lysine residues in site B were converted to glutamine to mimic acetylated lysines. The mutations significantly reduced HDAC6 tubulin deacetylase activity and further impaired cell motility, but had no effect on histone deacetylase activity. More interestingly, these mutations retained HDAC6 in the cytoplasm by blocking the interaction with the nuclear import protein importin-α. The retention of HDAC6 in the cytoplasm by acetylation eventually affects histone deacetylation. Thus, we conclude that acetylation is an important post-translational modification that regulates HDAC6 tubulin deacetylase activity and nuclear import.

Vitamin D status among residents of Molidawa Daur Autonomous Banner, Inner Mongolia, North China.

OBJECTIVES: To retrospectively analyze the vitamin D (VD) status of residents in northeastern Inner Mongolia and its relationship with the average monthly sunshine hours. METHODS: Serum 25-hydroxyvitamin D[s-25(OH)D] samples from 4982 outpatients (2092 males) in Moli Dawa Daur Autonomous Banner People's Hospital, Hulunbuir, China from July 2018 to January 2022 were included in this study. RESULTS: The overall median s-25(OH)D was 53.3 nmol/L, VD deficiency (<30 nmol/L), deficiency (30-50 nmol/L), sufficient (>50-250 nmol/L) and excess (>250 nmol/L) were 16% (796/4982), 30% (1495/4982), 53.4% (2658/4982) and 0.7% (33/4982). There were statistically significant differences in median s-25(OH)D by month, age-groups and gender (p<0.001). Low VD status (LVDS, including VD deficiency and insufficiency) in females was 54.6% and males was 33.9%, and the LVDS composition differed significantly by age-group and month (p<0.05). The changing trend of the median s-25(OH)D level was similar to the monthly average sunshine hours, with a slight lag. CONCLUSION: Nearly half of residents live in LVDS. LVDS is affected by month, gender, and age.

Vorinostat: a potent agent to prevent and treat laser-induced corneal haze.

PURPOSE: This study investigated the efficacy and safety of vorinostat, a deacetylase (HDAC) inhibitor, in the treatment of laser-induced corneal haze following photorefractive keratectomy (PRK) in rabbits in vivo and transforming growth factor beta 1 (TGFß1) -induced corneal fibrosis in vitro. METHODS: Corneal haze in rabbits was produced with -9.00 diopters (D) PRK. Fibrosis in cultured human and rabbit corneal fibroblasts was activated with TGFß1. Vorinostat (25 µm) was topically applied once for 5 minutes on rabbit cornea immediately after PRK for in vivo studies. Vorinostat (0 to 25 µm) was given to human/rabbit corneal fibroblasts for 5 minutes or 48 hours for in vitro studies. Slit-lamp microscopy, TUNEL assay, and trypan blue were used to determined vorinostat toxicity, whereas real-time polymerase chain reaction, immunocytochemistry, and immunoblotting were used to measure its efficacy. RESULTS: Single 5-minute vorinostat (25 µm) topical application on the cornea following PRK significantly reduced corneal haze (P<.008) and fibrotic marker proteins (α-smooth muscle actin and f-actin; P<.001) without showing redness, swelling, or inflammation in rabbit eyes in vivo screened 4 weeks after PRK. Vorinostat reduced TGFß1-induced fibrosis in human and rabbit corneas in vitro in a dose-dependent manner without altering cellular viability, phenotype, or proliferation. CONCLUSIONS: Vorinostat is non-cytotoxic and safe for the eye and has potential to prevent laser-induced corneal haze in patients undergoing PRK for high myopia.

Preclinical evaluation of WVE-004, aninvestigational stereopure oligonucleotide forthe treatment of C9orf72-associated ALS or FTD.

A large hexanucleotide (G4C2) repeat expansion in the first intronic region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several mechanisms have been proposed to explain how the repeat expansion drives disease, and we hypothesize that a variant-selective approach, in which transcripts affected by the repeat expansion are preferentially decreased, has the potential to address most of them. We report a stereopure antisense oligonucleotide, WVE-004, that executes this variant-selective mechanism of action. WVE-004 dose-dependently and selectively reduces repeat-containing transcripts in patient-derived motor neurons carrying a C9orf72-repeat expansion, as well as in the spinal cord and cortex of C9 BAC transgenic mice. In mice, selective transcript knockdown was accompanied by substantial decreases in dipeptide-repeat proteins, which are pathological biomarkers associated with the repeat expansion, and by preservation of healthy C9orf72 protein expression. These in vivo effects were durable, persisting for at least 6 months. These data support the advancement of WVE-004 as an investigational stereopure antisense oligonucleotide targeting C9orf72 for the treatment of C9orf72-associated ALS or FTD.

Variant-selective stereopure oligonucleotides protect against pathologies associated with C9orf72-repeat expansion in preclinical models.

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.

Behavior of Salmonella Typhimurium on Fresh Strawberries Under Different Storage Temperatures and Wash Treatments.

Fresh strawberries are one of the most popular fruits in China and are vulnerable to microbial contamination. In this study, the behavior of Salmonella Typhimurium on fresh strawberries stored at refrigeration and room temperatures, as well as the effectiveness of mild heat wash treatments at 47, 50, and 53°C on bacterial survival was investigated. The modified Gompertz, Huang, log-linear, and Weibull models were used to fit bacterial growth and survival curves under different treatments. A secondary model based on linear regression was developed to describe the effect of washing temperature on the kinetic parameters of S. Typhimurium survival derived from the Weibull model. During 72 h storage, S. Typhimurium on fresh strawberries stored at 4°C was reduced by 1.35 log CFU/g and growth of 5.64 log CFU/g was observed when strawberries were stored at 25°C. Bacterial reductions of 1.22 ± 0.15, 1.92 ± 0.06, 2.27 ± 0.07 log CFU/g were obtained when washing was carried out at 47, 50 and 53°C for 240 s, respectively. The wash temperature was an important parameter for bacterial inactivation and bacterial populations declined significantly in conjunction with washing time (p < 0.05). Warm wash treatments lead the visible color changes of strawberries, showing a slightly darker appearance while acceptable. The goodness-of-fit indices indicated that the log-linear model provided a satisfactory fit to describe the bacterial survival at 4°C. According to the smaller Akaike information criterion (AIC) value, the modified Gompertz model performed slightly better than the Huang model in describing bacterial growth at 25°C. The high adj-R2 (≥0.90) and small RMSE (≤0.22) indicated the Weibull model better described bacterial behavior under mild heat treatments. We found a close linear relationship between wash temperatures and ln k and ln n. These models were validated by independent experimental data and the values of the bias and accuracy factors fell into the acceptable range.

Evaluation of Sanitizing Methods for Reducing Microbial Contamination on Fresh Strawberry, Cherry Tomato, and Red Bayberry.

Strawberries, cherry tomatoes, and red bayberries, which are the most popular types of fresh produce in China, are vulnerable to microbial contamination. In this study, different sanitizing methods [treatment with 2% organic acids, 0.02% sodium hypochlorite (SH), 0.1% sodium chlorite (SC), and 0.1% acidified sodium chlorite (ASC)] were applied to fresh strawberry, cherry tomato, and red bayberry, and their abilities to reduce aerobic bacteria, Escherichia coli O157:H7, mold, yeast, and Salmonella Typhimurium were evaluated. The commercially used SH method reduced the background microbiota on strawberry, cherry tomato, and red bayberry by 0.20-2.07 log cfu/g. The ASC method reduced background microbiota (except for mold) on strawberry and cherry tomato by more than 3.0 log cfu/g. ASC was the only sanitizer that significantly reduced mold on red bayberry, and lactic acid was the only organic acid sanitizer that effectively reduced yeast on red bayberry. The ASC method had the best sterilizing effect on the three fresh fruits and also required the shortest sanitizing time and low chlorite content. The application of ASC method significantly reduced the microbiota on retail grocery samples, and the effect was similar to that achieved by sanitizing methods comparison.

RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2.

Several microsatellite-expansion diseases are characterized by the accumulation of RNA foci and RAN proteins, raising the possibility of a mechanistic connection. We explored this question using myotonic dystrophy type 2, a multisystemic disease thought to be primarily caused by RNA gain-of-function effects. We demonstrate that the DM2 CCTG⋅CAGG expansion expresses sense and antisense tetrapeptide poly-(LPAC) and poly-(QAGR) RAN proteins, respectively. In DM2 autopsy brains, LPAC is found in neurons, astrocytes, and glia in gray matter, and antisense QAGR proteins accumulate within white matter. LPAC and QAGR proteins are toxic to cells independent of RNA gain of function. RNA foci and nuclear sequestration of CCUG transcripts by MBNL1 is inversely correlated with LPAC expression. These data suggest a model that involves nuclear retention of expansion RNAs by RNA-binding proteins (RBPs) and an acute phase in which expansion RNAs exceed RBP sequestration capacity, are exported to the cytoplasm, and undergo RAN translation. VIDEO ABSTRACT.

C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD.

To define how the C9orf72 GGGGCC expansion mutation causes ALS/FTD and to facilitate therapy development, a mouse model that recapitulates the molecular and phenotypic features of the disease is urgently needed. Two groups recently reported BAC mouse models that produce RNA foci and RAN proteins but, surprisingly, do not develop the neurodegenerative or behavioral features of ALS/FTD. We now report a BAC mouse model of C9orf72 ALS/FTD that shows decreased survival, paralysis, muscle denervation, motor neuron loss, anxiety-like behavior, and cortical and hippocampal neurodegeneration. These mice express C9orf72 sense transcripts and upregulated antisense transcripts. In contrast to sense RNA foci, antisense foci preferentially accumulate in ALS/FTD-vulnerable cell populations. RAN protein accumulation increases with age and disease, and TDP-43 inclusions are found in degenerating brain regions in end-stage animals. The ALS/FTD phenotypes in our mice provide a unique tool that will facilitate developing therapies targeting pathways that prevent neurodegeneration and increase survival.