Intragenic complementation of amino and carboxy terminal SMN missense mutations can rescue Smn null mice.

Spinal muscular atrophy is caused by reduced levels of SMN resulting from the loss of SMN1 and reliance on SMN2 for the production of SMN. Loss of SMN entirely is embryonic lethal in mammals. There are several SMN missense mutations found in humans. These alleles do not show partial function in the absence of wild-type SMN and cannot rescue a null Smn allele in mice. However, these human SMN missense allele transgenes can rescue a null Smn allele when SMN2 is present. We find that the N- and C-terminal regions constitute two independent domains of SMN that can be separated genetically and undergo intragenic complementation. These SMN protein heteromers restore snRNP assembly of Sm proteins onto snRNA and completely rescue both survival of Smn null mice and motor neuron electrophysiology demonstrating that the essential functional unit of SMN is the oligomer.

Mild SMN missense alleles are only functional in the presence of SMN2 in mammals.

Spinal muscular atrophy (SMA) is caused by reduced levels of full-length SMN (FL-SMN). In SMA patients with one or two copies of the Survival Motor Neuron 2 (SMN2) gene there are a number of SMN missense mutations that result in milder-than-predicted SMA phenotypes. These mild SMN missense mutation alleles are often assumed to have partial function. However, it is important to consider the contribution of FL-SMN as these missense alleles never occur in the absence of SMN2. We propose that these patients contain a partially functional oligomeric SMN complex consisting of FL-SMN from SMN2 and mutant SMN protein produced from the missense allele. Here we show that mild SMN missense mutations SMND44V, SMNT74I or SMNQ282A alone do not rescue mice lacking wild-type FL-SMN. Thus, missense mutations are not functional in the absence of FL-SMN. In contrast, when the same mild SMN missense mutations are expressed in a mouse containing two SMN2 copies, functional SMN complexes are formed with the small amount of wild-type FL-SMN produced by SMN2 and the SMA phenotype is completely rescued. This contrasts with SMN missense alleles when studied in C. elegans, Drosophila and zebrafish. Here we demonstrate that the heteromeric SMN complex formed with FL-SMN is functional and sufficient to rescue small nuclear ribonucleoprotein assembly, motor neuron function and rescue the SMA mice. We conclude that mild SMN missense alleles are not partially functional but rather they are completely non-functional in the absence of wild-type SMN in mammals.

An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody.

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.

Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector.

Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood-brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES: Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS: Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS: Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION: High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Intramuscular scAAV9-SMN injection mediates widespread gene delivery to the spinal cord and decreases disease severity in SMA mice.

We have recently demonstrated the remarkable efficiency of self-complementary (sc) AAV9 vectors for central nervous system (CNS) gene transfer following intravenous delivery in mice and larger animals. Here, we investigated whether gene delivery to motor neurons (MNs) could also be achieved via intramuscular (i.m.) scAAV9 injection and subsequent retrograde transport along the MNs axons. Unexpectedly, we found that a single injection of scAAV9 into the adult mouse gastrocnemius (GA) mediated widespread MN transduction along the whole spinal cord, without limitation to the MNs connected to the injected muscle. Spinal cord astrocytes and peripheral organs were also transduced, indicating vector spread from the injected muscle to both the CNS and the periphery through release into the blood circulation. Moreover, we showed that i.m. injection of scAAV9 vectors expressing "survival of motor neuron" (Smn) in spinal muscular atrophy (SMA) mice mediated high survival motor neuron (SMN) expression levels at both the CNS and the periphery, and increased the median lifespan from 12 days to 163 days. These findings represent to date the longest extent in survival obtained in SMA mice following i.m. viral vector gene delivery, and might generate a renewed interest in the use of i.m. adeno-associated viruses (AAV) delivery for the development of gene therapy strategies for MN diseases.

Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice.

Spinal muscular atrophy (SMA) is the most common genetic disease leading to infant mortality. This neuromuscular disorder is caused by the loss or mutation of the telomeric copy of the 'survival of motor neuron' (Smn) gene, termed SMN1. Loss of SMN1 leads to reduced SMN protein levels, inducing degeneration of motor neurons (MN) and progressive muscle weakness and atrophy. To date, SMA remains incurable due to the lack of a method to deliver therapeutically active molecules to the spinal cord. Gene therapy, consisting of reintroducing SMN1 in MNs, is an attractive approach for SMA. Here we used postnatal day 1 systemic injection of self-complementary adeno-associated virus (scAAV9) vectors carrying a codon-optimized SMN1 sequence and a chimeric intron placed downstream of the strong phosphoglycerate kinase (PGK) promoter (SMNopti) to overexpress the human SMN protein in a mouse model of severe SMA. Survival analysis showed that this treatment rescued 100% of the mice, increasing life expectancy from 27 to over 340 days (median survival of 199 days) in mice that normally survive about 13 days. The systemic scAAV9 therapy mediated complete correction of motor function, prevented MN death and rescued the weight loss phenotype close to normal. This study reports the most efficient rescue of SMA mice to date after a single intravenous injection of an optimized SMN-encoding scAAV9, highlighting the considerable potential of this method for the treatment of human SMA.

Reduced dengue incidence following city-wide wMel Wolbachia mosquito releases throughout three Colombian cities: Interrupted time series analysis and a prospective case-control study.

BACKGROUND: The introduction of Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Randomised and non-randomised studies in multiple countries have shown significant reductions in dengue incidence following field releases of wMel-infected Ae. aegypti. We report the public health outcomes from phased, large-scale releases of wMel-Ae. aegypti mosquitoes throughout three contiguous cities in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Following pilot releases in 2015-2016, staged city-wide wMel-Ae. aegypti deployments were undertaken in the cities of Bello, Medellín and Itagüí (3.3 million people) between October 2016 and April 2022. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study using interrupted time series analysis showed notified dengue case incidence was reduced by 95% in Bello and Medellín and 97% in Itagüí, following establishment of wMel at ≥60% prevalence, compared to the pre-intervention period and after adjusting for seasonal trends. A concurrent clinic-based case-control study with a test-negative design was unable to attain the target sample size of 63 enrolled virologically-confirmed dengue (VCD) cases between May 2019 and December 2021, consistent with low dengue incidence throughout the Aburrá Valley following wMel deployments. Nevertheless, VCD incidence was 45% lower (OR 0.55 [95% CI 0.25, 1.17]) and combined VCD/presumptive dengue incidence was 47% lower (OR 0.53 [95% CI 0.30, 0.93]) among participants resident in wMel-treated versus untreated neighbourhoods. CONCLUSIONS/SIGNIFICANCE: Stable introduction of wMel into local Ae. aegypti populations was associated with a significant and sustained reduction in dengue incidence across three Colombian cities. These results from the largest contiguous Wolbachia releases to-date demonstrate the real-world effectiveness of the method across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings. TRIAL REGISTRATION: NCT03631719.

Tislelizumab in Patients with Previously Treated Advanced Hepatocellular Carcinoma (RATIONALE-208): A Multicenter, Non-Randomized, Open-Label, Phase 2 Trial.

Introduction: Tislelizumab (anti-programmed cell death protein 1 antibody) showed preliminary antitumor activity and tolerability in patients with advanced solid tumors, including hepatocellular carcinoma (HCC). This study aimed to assess the efficacy and safety of tislelizumab in patients with previously treated advanced HCC. Methods: The multiregional phase 2 study RATIONALE-208 examined single-agent tislelizumab (200 mg intravenously every 3 weeks) in patients with advanced HCC with Child-Pugh A, Barcelona Clinic Liver Cancer stage B or C, and who had received one or more prior lines of systemic therapy. The primary endpoint was objective response rate (ORR), radiologically confirmed per Response Evaluation Criteria in Solid Tumors version 1.1 by the Independent Review Committee. Safety was assessed in patients who received ≥1 dose of tislelizumab. Results: Between April 9, 2018, and February 27, 2019, 249 eligible patients were enrolled and treated. After a median study follow-up of 12.7 months, ORR was 13% (n = 32/249; 95% confidence interval [CI], 9-18), including five complete and 27 partial responses. The number of prior lines of therapy did not impact ORR (one prior line, 13% [95% CI, 8-20]; two or more prior lines, 13% [95% CI, 7-20]). Median duration of response was not reached. The disease control rate was 53%, and median overall survival was 13.2 months. Of the 249 total patients, grade ≥3 treatment-related adverse events were reported in 38 (15%) patients; the most common was liver transaminase elevations in 10 (4%) patients. Treatment-related adverse events led to treatment discontinuation in 13 (5%) patients or dose delay in 46 (19%) patients. No deaths were attributed to the treatment per investigator assessment. Conclusion: Tislelizumab demonstrated durable objective responses, regardless of the number of prior lines of therapy, and acceptable tolerability in patients with previously treated advanced HCC.

Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders.

Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood-brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations.

AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Phenolic derivatives from Ruprechtia polystachya and their inhibitory activities on the glucose-6-phosphatase system.

Two new compounds, 5-methyl-2-(2-methylbutanoyl)phloroglucinol 1-O-(6-O-ß-D-apiofuranosyl)-ß-D-glucopyranoside (1) and trans-2,3-dihydrokaempferol 3-O-(4-O-sulfo)-α-L-arabinopyranoside (2), together with 14 known flavonoids, trans-dihydrokaempferol 3-O-α-L-arabinopyranoside (3), trans-taxifolin 3-O-α-L-arabinofuranoside (4), quercetin 3-O-α-L-rhamnopyranoside (5), quercetin 3'-O-α-L-arabinofuranoside (6), catechin 3-O-α-L-rhamnopyranoside (7), trans-taxifolin 3-O-α-L-arabinopyranoside (8), cis-dihydrokaempferol 3-O-α-L-arabinopyranoside (9), catechin (10), myricetin 3-O-α-L-rhamnopyranoside (11), quercetin 3-O-α-L-arabinopyranoside (12), quercetin 3-O-α-L-arabinofuranoside (13), quercetin 3-O-(3″-galloyl)-α-L-rhamnopyranoside (14), quercetin 3-O-(2″-galloyl)-α-L-rhamnopyranoside (15), and epicatechin 3-O-gallate (16), were isolated from the leaves of Ruprechtia polystachya Griseb. (Polygonaceae). Their structures were established on the basis of extensive 1D- and 2D-NMR experiments as well as MS analyses. All compounds, except 1, showed inhibition of the enzyme glucose-6-phosphatase in intact microsomes.

AAV9-mediated gene delivery of MCT1 to oligodendrocytes does not provide a therapeutic benefit in a mouse model of ALS.

Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1G93A mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1G93A mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells.

Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.

Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood-brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients.

The impact of city-wide deployment of Wolbachia-carrying mosquitoes on arboviral disease incidence in Medellín and Bello, Colombia: study protocol for an interrupted time-series analysis and a test-negative design study.

Background: Dengue, chikungunya and Zika are viral infections transmitted by Aedes aegypti mosquitoes, and present major public health challenges in tropical regions. Traditional vector control methods have been ineffective at halting disease transmission. The World Mosquito Program has developed a novel approach to arbovirus control using Ae. aegypti stably transfected with the Wolbachia bacterium, which have significantly reduced ability to transmit dengue, Zika and chikungunya in laboratory experiments. Field releases in eight countries have demonstrated Wolbachia establishment in local Ae. aegypti populations. Methods: We describe a pragmatic approach to measuring the epidemiological impact of city-wide Wolbachia deployments in Bello and Medellín, Colombia. First, an interrupted time-series analysis will compare the incidence of dengue, chikungunya and Zika case notifications before and after Wolbachia releases, across the two municipalities. Second, a prospective case-control study using a test-negative design will be conducted in one quadrant of Medellín. Three of the six contiguous release zones in the case-control area were allocated to receive the first Wolbachia deployments in the city and three to be treated last, approximating a parallel two-arm trial for the >12-month period during which Wolbachia exposure remains discordant. Allocation, although non-random, aimed to maximise balance between arms in historical dengue incidence and demographics. Arboviral disease cases and arbovirus-negative controls will be enrolled concurrently from febrile patients presenting to primary care, with case/control status classified retrospectively following laboratory diagnostic testing. Intervention effect is estimated from an aggregate odds ratio comparing Wolbachia-exposure odds among test-positive cases versus test-negative controls. Discussion: The study findings will add to an accumulating body of evidence from global field sites on the efficacy of the Wolbachia method in reducing arboviral disease incidence, and can inform decisions on wider public health implementation of this intervention in the Americas and beyond. Trial registration: ClinicalTrials.gov: NCT03631719. Registered on 15 August 2018.

SMN Blood Levels in a Porcine Model of Spinal Muscular Atrophy.

BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal recessive motor neuron disease that results in loss of spinal motor neurons, muscular weakness and, in severe cases, respiratory failure and death. SMA is caused by a deletion or mutation of the SMN1 gene and retention of the SMN2 gene that leads to low SMN expression levels.The measurement of SMN mRNA levels in peripheral blood samples has been used in SMA clinical studies as a pharmacodynamic biomarker for response to therapies designed to increase SMN levels. We recently developed a postnatal porcine model of SMA by the viral delivery of a short-hairpin RNA (shRNA) targeting porcine SMN (pSMN). scAAV9-mediated knockdown of pSMN mRNA at postnatal day 5 results in denervation, weakness and motor neuron and ventral root axon loss that begins 3-4 weeks after viral delivery, and this phenotype can be ameliorated by subsequent viral delivery of human SMN (hSMN). OBJECTIVE: To determine if the effect of modulating SMN levels using gene therapy can be measured in blood. METHODS: We measured expression of pSMN mRNA and hSMN mRNA by quantitative droplet digital PCR (ddPCR). RESULTS: We found that the endogenous expression of pSMN mRNA in blood increases in the first month of life. However, there were no significant differences in blood levels of pSMN mRNA after knock-down or of human SMN mRNA after gene therapy. CONCLUSIONS: Our results, obtained in a large animal model of SMA that is similar in size and anatomy to human infants, suggest that measurement of SMN mRNA levels in blood may not be informative in SMA clinical trials involving intrathecal delivery of SMN-modulating therapies.

Esfuerzos generados en la ATM en maloclusión clase II, tratada con exodoncias de premolares y ortodoncia: análisis mediante el método de elementos finitos / Strain generated in the TMJ with class II malocclusions, treated with extraction of premolars and orthodontics: analysis with the finite element method

RESUMEN Introducción: la exodoncia de premolares es una alternativa para el tratamiento de la maloclusión clase II. Un cambio en la biomecánica puede generar alteraciones en la Articulación Temporomandibular (ATM) lo que produce mayor desgaste dental y aparición de patologías articulares. El objetivo fue analizar mediante el método de elementos finitos la concentración de esfuerzos en la ATM, en maloclusión clase II, tratados con exodoncia de premolares y Ortodoncia. Métodos: dos modelos de simulación en 3D cada uno con estructuras óseas de los 2 maxilares, dentición completa y disco en la ATM. Uno corresponde al paciente sin recidiva (SR) tratado con exodoncia de primeros premolares y ortodoncia, donde se mantiene la estabilidad dental clase I. El otro modelo con recidiva (CR) tratada con exodoncia de primeros premolares y ortodoncia, aumento de overjet y overbite y clase II canina; la carga se aplicó sobre la rama mandibular. Resultados: con una carga de 900N los esfuerzos se triplicaron en todas las estructuras de los dos modelos al ser comparados con una carga de 300N; sin embargo, se dieron diferencias considerables en el modelo CR entre las cavidades glenoideas, a 300N de 19.9 MPa y a 900N de 59.3 MPa. La mayor concentración del disco se da en la parte lateral. Conclusiones: dada la asimetría en las estructuras de la ATM, los esfuerzos y la concentración de tensiones difieren entre el lado derecho e izquierdo en los dos modelos.

Abstract Introduction: premolar extraction is an alternative for the treatment of class II malocclusion. A change in biomechanics can generate alterations in the Temporomandibular Joint (TMJ), which produces greater dental wear and the appearance of joint dysfunctions. The objective was to assess the effort concentration in the TMJ by means of finite element analysis in class II malocclusions treated with premolar extraction and orthodontics. Method: two 3D simulation models each with bone structures of the 2 jaws, complete dentition and disc in the TMJ. One corresponds to the patient without recurrence (WR) treated with extraction of first premolars and orthodontics, where class I dental stability is maintained. The other model with recurrence (R) treated with extraction of first premolars and orthodontics, increased overjet and overbite and canine class II; the load was applied to the mandibular ramus. Results: loads of 900N triplicated on all structures compared to 300N in both models. However, there were considerable differences between the left and right glenoid cavities in the WR model, at 300N of 19.9 MPa and 900N at 59.3 MPa. Most tensions of the disc occur in the lateral part. Conclusions: due to the asymmetry in the TMJ structures, the stresses and stress concentration differ between the right and left sides in the two models.

Normalization of Patient-Identified Plasma Biomarkers in SMNΔ7 Mice following Postnatal SMN Restoration.

INTRODUCTION AND OBJECTIVE: Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder. SMA is caused by homozygous loss of the SMN1 gene and retention of the SMN2 gene resulting in reduced levels of full length SMN protein that are insufficient for motor neuron function. Various treatments that restore levels of SMN are currently in clinical trials and biomarkers are needed to determine the response to treatment. Here, we sought to investigate in SMA mice a set of plasma analytes, previously identified in patients with SMA to correlate with motor function. The goal was to determine whether levels of plasma markers were altered in the SMNΔ7 mouse model of SMA and whether postnatal SMN restoration resulted in normalization of the biomarkers. METHODS: SMNΔ7 and control mice were treated with antisense oligonucleotides (ASO) targeting ISS-N1 to increase SMN protein from SMN2 or scramble ASO (sham treatment) via intracerebroventricular injection on postnatal day 1 (P1). Brain, spinal cord, quadriceps muscle, and liver were analyzed for SMN protein levels at P12 and P90. Ten plasma biomarkers (a subset of biomarkers in the SMA-MAP panel available for analysis in mice) were analyzed in plasma obtained at P12, P30, and P90. RESULTS: Of the eight plasma biomarkers assessed, 5 were significantly changed in sham treated SMNΔ7 mice compared to control mice and were normalized in SMNΔ7 mice treated with ASO. CONCLUSION: This study defines a subset of the SMA-MAP plasma biomarker panel that is abnormal in the most commonly used mouse model of SMA. Furthermore, some of these markers are responsive to postnatal SMN restoration. These findings support continued clinical development of these potential prognostic and pharmacodynamic biomarkers.

Electrophysiological Biomarkers in Spinal Muscular Atrophy: Preclinical Proof of Concept.

OBJECTIVE: Preclinical therapies that restore survival motor neuron (SMN) protein levels can dramatically extend survival in spinal muscular atrophy (SMA) mouse models. Biomarkers are needed to effectively translate these promising therapies to clinical trials. Our objective was to investigate electrophysiological biomarkers of compound muscle action potential (CMAP), motor unit number estimation (MUNE) and electromyography (EMG) using an SMA mouse model. METHODS: Sciatic CMAP, MUNE, and EMG were obtained in SMNΔ7 mice at ages 3-13 days and at 21 days in mice with SMN selectively reduced in motor neurons (ChATCre ). To investigate these measures as biomarkers of treatment response, measurements were obtained in SMNΔ7 mice treated with antisense oligonucleotide (ASO) or gene therapy. RESULTS: CMAP was significantly reduced in SMNΔ7 mice at days 6-13 (p<0.01), and MUNE was reduced at days 7-13 (p<0.01). Fibrillations were present on EMG in SMNΔ7 mice but not controls (p=0.02). Similar findings were seen at 21 days in ChATCre mice. MUNE in ASO-treated SMNΔ7 mice were similar to controls at day 12 and 30. CMAP reduction persisted in ASO-treated SMNΔ7 mice at day 12 but was corrected at day 30. Similarly, CMAP and MUNE responses were corrected with gene therapy to restore SMN. INTERPRETATION: These studies confirm features of preserved neuromuscular function in the early postnatal period and subsequent motor unit loss in SMNΔ7 mice. SMN restoring therapies result in preserved MUNE and gradual repair of CMAP responses. This provides preclinical evidence for the utilization of CMAP and MUNE as biomarkers in future SMA clinical trials.

Orthodontic treatment stability and periodontal condition with circumferential supracrestal fiberotomy: a systematic review / Estabilidad del tratamiento de ortodoncia y de la condición periodontal con la fibrotomía supracrestal circunferencial: revisión sistemática

ABSTRACT Introduction: stability is one of the main goals of orthodontic treatment, and circumferential supracrestal fiberotomy is an alternative to prevent relapse in cases of tooth rotation, crowding and inclined teeth. However, there are no studies demonstrating the effectiveness of this treatment and its effects on the periodontal condition. The aim of this systematic review (SR) was to evaluate the effectiveness of circumferential supracrestal fiberotomy (CSF) as an adjuvant in the stability of orthodontic treatment during retention and its effects on the periodontal condition once it has been performed. Methods: the search for topic-related studies was conducted on the PubMed and EMBASE databases until October 2018. The studies were considered eligible if they covered the use of CSF during the retention period and reported the periodontal condition in a follow-up period longer than or equal to 1 year. For bias-risk assessment in the chosen studies, the Newcastle-Ottawa Scale was applied to observational studies, and the Cochrane Collaboration tool for Randomized Clinical Trials (RCTs) and Controlled Clinical Trials (CCTs). Results: the search strategy yielded 85 potential eligible articles, of which 5 were included in the SR. Four of the five studies reported a lower irregularity rate in patients who had CSF when compared to a control group. No changes in plaque index, gingival index, insertion levels, probe depth and keratinized gingiva amount were reported. Conclusions: fiberotomy is an effective method to prevent relapse of previously rotated teeth and does not cause periodontal alterations. However, it is important to note that the studies' methodological quality was low.

RESUMEN Introducción: la estabilidad es uno de los principales objetivos del tratamiento de ortodoncia, y la fibrotomía supracrestal circunferencial es una de las alternativas para prevenir la recidiva en casos de rotaciones dentales, apiñamiento y dientes con inclinación; sin embargo, no se tienen estudios que demuestren la efectividad de este tratamiento, así como sus efectos en la condición periodontal. El objetivo de esta revisión sistemática (RS) consistió en evaluar la efectividad de la fibrotomía supracrestal circunferencial (FSC) como procedimiento coadyuvante en la estabilidad del tratamiento de ortodoncia durante la retención, así como los efectos en la condición periodontal posterior a su realización. Métodos: la búsqueda de estudios relacionados con el tema se realizó mediante las bases de datos PubMed y EMBASE hasta octubre de 2018. Los estudios fueron considerados elegibles si abarcaban el uso de la FSC durante el periodo de retención y si reportaban la condición periodontal con un tiempo de seguimiento mayor o igual a un año. Para la evaluación del riesgo de sesgos en los estudios elegidos, se aplicó la escala Newcastle-Ottawa en los estudios observacionales y la herramienta de colaboración Cochrane para los ensayos clínicos aleatorizados (ECA) y ensayos clínicos controlados (ECC). Resultados: la estrategia de búsqueda arrojó 85 posibles artículos elegibles, de los cuales 5 fueron incluidos en la RS. Cuatro de los cinco estudios reportaron un índice de irregularidad menor en los pacientes que tuvieron FSC cuando se compararon con un grupo control. Con respecto a la condición periodontal, no se reportaron cambios en índice de placa, índice gingival, niveles de inserción, profundidad al sondaje y cantidad de encía queratinizada. Conclusiones: la fibrotomía es un método eficaz para evitar la recidiva de dientes previamente rotados y no genera alteraciones a nivel periodontal. Sin embargo, es importante tener en cuenta que la calidad metodológica de los estudios no fue alta.