EZcalcium: Open-Source Toolbox for Analysis of Calcium Imaging Data.

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted "miniscopes," is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P, and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium.

Author Correction: Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible.

The original and corrected Acknowledgements are shown in the accompanying Author Correction.

Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible.

To uncover the circuit-level alterations that underlie atypical sensory processing associated with autism, we adopted a symptom-to-circuit approach in the Fmr1-knockout (Fmr1-/-) mouse model of Fragile X syndrome. Using a go/no-go task and in vivo two-photon calcium imaging, we find that impaired visual discrimination in Fmr1-/- mice correlates with marked deficits in orientation tuning of principal neurons and with a decrease in the activity of parvalbumin interneurons in primary visual cortex. Restoring visually evoked activity in parvalbumin cells in Fmr1-/- mice with a chemogenetic strategy using designer receptors exclusively activated by designer drugs was sufficient to rescue their behavioral performance. Strikingly, human subjects with Fragile X syndrome exhibit impairments in visual discrimination similar to those in Fmr1-/- mice. These results suggest that manipulating inhibition may help sensory processing in Fragile X syndrome.

Soluble tau aggregates, not large fibrils, are the toxic species that display seeding and cross-seeding behavior.

Several studies have proposed that fibrillary aggregates of tau and other amyloidogenic proteins are neurotoxic and result in numerous neurodegenerative diseases. However, these studies usually involve sonication or extrusion through needles before experimentation. As a consequence, these methods may fragment large aggregates producing a mixture of aggregated species rather than intact fibrils. Therefore, the results of these experiments may be reflective of other amyloidogenic species, such as oligomers and/or protofibrils/short fibrils. To investigate the effects of sonication on the aggregation of tau and other amyloidogenic proteins, fibrils were prepared and well characterized, then sonicated and evaluated by various biochemical and biophysical methods to identify the aggregated species present. We found that indeed a mixture of aggregated species was present along with short fibrils indicating that sonication leads to impure fibril samples and should be analyzed with caution. Our results corroborate the previous studies showing that sonication of prion and Aß fibrils leads to the formation of toxic, soluble aggregates. We also show that the oligomeric forms are the most toxic species although it is unclear how sonication causes oligomer formation. Recent results suggest that these small toxic oligomers produced by sonication, rather than the stable fibrillar structures, are prion-like in nature displaying seeding and cross-seeding behavior.

A Versatile Method for Viral Transfection of Calcium Indicators in the Neonatal Mouse Brain.

The first three postnatal weeks in rodents are a time when sensory experience drives the maturation of brain circuits, an important process that is not yet well understood. Alterations in this critical period of experience-dependent circuit assembly and plasticity contribute to several neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. Therefore, techniques for recording network activity and tracing neuronal connectivity over this time period are necessary for delineating circuit refinement in typical development and how it deviates in disease. Calcium imaging with GCaMP6 and other genetically encoded indicators is rapidly becoming the preferred method for recording network activity at the single-synapse and single-cell level in vivo, especially in genetically identified neuronal populations. We describe a protocol for intracortical injection of recombinant adeno-associated viruses in P1 neonatal mice and demonstrate its use for longitudinal imaging of GCaMP6s in the same neurons over several weeks to characterize the developmental desynchronization of cortical network activity. Our approach is ideally suited for chronic in vivo two-photon calcium imaging of neuronal activity from synapses to entire networks during the early postnatal period.

[Recommendations for the use of ranibizumab in diabetic macular edema at IMSS]. / Recomendaciones para el uso de ranibizumab en edema macular diabético en el IMSS.

Diabetic macular edema can occur at any stage of diabetic retinopathy. It represents the main cause of vision loss in diabetes type I and II with a prevalence of 3-10% in diabetic patients of the Instituto Mexicano del Seguro Social (IMSS). Our aim is to elaborate treatment guidelines and provide recommendations for the use of intravitreal ranibizumab for diabetic medical edema at IMSS. Nine retina specialists and 10 ophthalmologists from IMSS high specialty medical units gathered to discuss the bibliographic evidence for the safety and efficacy of ranibizumab for this disease, in order to create consensus on its use in the institution. Intravitreal ranibizumab injection should be used on patients presenting diffuse or cystic diabetic macular edema who have strict metabolic control and visual acuity between 20/30 and 20/200 ETDRS, as well as structural features, such as inferior foveal limit of 280 µm and ischemic areas no larger than 50% of the central foveal area. Treatment regime should consist of a loading charge of three monthly injections of ranibizumab 0.5 mg, followed by monthly follow-ups and treatment as needed according to anatomic and functional criteria. This consensus decision-making process on the criteria to treat and re-treat patients with this drug will result in better health outcomes than those currently observed among patients with diabetic macular edema at IMSS.

El edema macular diabético se presenta en cualquier etapa de la retinopatía diabética y representa la principal causa de pérdida de visión en las diabetes tipo I y II, con una prevalencia que va del 3 al 10% en pacientes diabéticos del Instituto Mexicano del Seguro Social (IMSS). El objetivo de este trabajo es elaborar una guía de tratamiento y recomendaciones para el uso de ranibizumab intravítreo en pacientes con edema macular diabético en el IMSS. Se llevó a cabo una reunión de expertos (9 retinólogos y 10 oftalmólogos) de las unidades médicas de alta especialidad del IMSS para realizar una revisión crítica de la eficacia y seguridad del ranibizumab para esta enfermedad y llegar a un consenso sobre el uso de este antiangiogénico en la institución. Las inyecciones de ranibizumab intravítreo se aplicarían a pacientes con edema macular diabético del tipo difuso o quístico, con un control metabólico estricto, agudeza visual en un rango de 20/30 a 20/200 ETDRS y criterios estructurales, como el límite foveal inferior a 280 µm y zonas isquémicas de no más del 50% de la zona central foveal. El esquema de tratamiento consistiría en una dosis de carga de tres inyecciones mensuales de ranibizumab de 0.5 mg y posteriormente seguimiento mensual y tratamiento por razón necesaria según criterios anatómicos y funcionales. El consenso sobre los criterios de tratamiento y retratamiento con este medicamento garantizará mejores resultados clínicos en pacientes con edema macular diabético en el IMSS.

Tactile Defensiveness and Impaired Adaptation of Neuronal Activity in the Fmr1 Knock-Out Mouse Model of Autism.

Sensory hypersensitivity is a common symptom in autism spectrum disorders (ASDs), including fragile X syndrome (FXS), and frequently leads to tactile defensiveness. In mouse models of ASDs, there is mounting evidence of neuronal and circuit hyperexcitability in several brain regions, which could contribute to sensory hypersensitivity. However, it is not yet known whether or how sensory stimulation might trigger abnormal sensory processing at the circuit level or abnormal behavioral responses in ASD mouse models, especially during an early developmental time when experience-dependent plasticity shapes such circuits. Using a novel assay, we discovered exaggerated motor responses to whisker stimulation in young Fmr1 knock-out (KO) mice (postnatal days 14-16), a model of FXS. Adult Fmr1 KO mice actively avoided a stimulus that was innocuous to wild-type controls, a sign of tactile defensiveness. Using in vivo two-photon calcium imaging of layer 2/3 barrel cortex neurons expressing GCaMP6s, we found no differences between wild-type and Fmr1 KO mice in overall whisker-evoked activity, though 45% fewer neurons in young Fmr1 KO mice responded in a time-locked manner. Notably, we identified a pronounced deficit in neuronal adaptation to repetitive whisker stimulation in both young and adult Fmr1 KO mice. Thus, impaired adaptation in cortical sensory circuits is a potential cause of tactile defensiveness in autism.SIGNIFICANCE STATEMENT We use a novel paradigm of repetitive whisker stimulation and in vivo calcium imaging to assess tactile defensiveness and barrel cortex activity in young and adult Fmr1 knock-out mice, the mouse model of fragile X syndrome (FXS). We describe evidence of tactile defensiveness, as well as a lack of L2/3 neuronal adaptation in barrel cortex, during whisker stimulation. We propose that a defect in sensory adaptation within local neuronal networks, beginning at a young age and continuing into adulthood, likely contributes to sensory overreactivity in FXS and perhaps other ASDs.

[Bone cement implant as an alternative for orbital floor reconstruction: A case report]. / Implante de cemento óseo como alternativa para la reconstrucción de piso orbitario: reporte de caso.

BACKGROUND: The management of orbitary fractures is one of the most challenging in facial trauma; the variety of reconstruction materials for its treatment is broad and is constantly improving, but despite this there is no consensus for its use or literature that sustains it. OBJECTIVE: To present the use and design of a preformed bone implant as an alternative for the reconstruction of orbital floor fractures in the pediatric age group. CLINICAL CASE: A 7-year old male who suffered a right hemifacial contusion trauma with clinical and tomographic diagnosis of right pure blowout type orbital floor fracture with inferior rectus muscle entrapment and right post-traumatic palpebral ptosis. Successful surgical reconstruction was performed 7 days later with a pre-constructed bone cement implant. Eight weeks after surgery the patient presented with mild residual palpebral ptosis, no ocular movement limitations and no diplopia. CONCLUSIONS: The use of a bone cement implant can be considered appropriate for the reconstruction of these fractures, as another alternative to be used by the ophthalmologist among the variety of all the other materials used for this purpose. We consider that our optimism based on the results obtained in this case obligates us to increase the number of patients treated in order to gather more evidence and do larger follow up.

Asociación de la hemorragia prerretiniana con la presencia y la gravedad de la retinopatía del prematuro en pacientes de alto riesgo.

OBJECTIVE: To assess whether preretinal hemorrhage (PRH) is associated with the presence and severity of retinopathy of prematurity (ROP) in high-risk patients. METHOD: Prospective cohort study, patients referred to the Department of Ophthalmology for ROP screening during October-November 2016 were evaluated weekly on 4 occasions to assess the relationship with PRH and the development of ROP as well as degree of severity associated. We used absolute, median frequencies with minimum and maximum values, χ2 test and Mann-Whitney U-test, as well as relative risk with 95% confidence interval. RESULTS: A total of 30 patients, in the first week 11 females (36%) and 4 males (13%) had PRH; in the 2nd week 13 patients (43%) presented PRH and 14 (46%) developed ROP; on the 3rd week there were no changes; in the last week 8 presented HPR (26%) and 11 patients (36%) with ROP. Stage I severity occurred more frequently. Statistical significance (p = 0.040) was found in the presence of PRH and the development of ROP in the first week with. CONCLUSIONS: HPR is a risk factor for the development of ROP during the first weeks of life and is associated with stages of mild severity.

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

[Effectiveness of trabeculectomy trabeculotomy in the reduction of intraocular pressure in patients with primary congenital glaucoma]. / Efectividad de la trabeculotomía más trabeculectomía en la reducción de la presión intraocular (PIO) en pacientes con glaucoma congénito primario (GCP).

OBJECTIVE: To evaluate the effectiveness of trabeculectomy trabeculotomy in the reduction of intraocular pressure in patients with primary congenital glaucoma. MATERIAL AND METHODS: A pre-experimental before and after study was conducted with several measurements after; patients with intraocular pressure≥21 mmHg were included, of both sexes, and children under six years, which had ​​the trabeculotomy trabeculectomy. Success was defined as the reduction of intraocular pressure<21 mmHg within three months after the procedure. For the descriptive analysis, medians with ranges and quartiles 25 and 75 were used. For the inferential analysis, we conducted a visual graphic and analysis of variance for repeated measures of Friedman. A value of p<0.05 was considered as significant. The software used was SPSSv15. RESULTS: 16 eyes were included with intraocular pressure 27.75 mmHg (23-40), vertical and horizontal corneal diameter of 14 mm (12-16), who had undergone trabeculectomy with mitomycin C trabeculectomy; at three months after surgery the intraocular pressure was 12.5 mmHg (8.5-23) (p<0.001) and no changes were observed in the corneal diameters. An eye with a flat anterior chamber was observed as an adverse effect. Thirteen eyes required topical hypotensors to achieve the target intraocular pressure. CONCLUSIONS: The treatment with trabeculectomy trabeculotomy in primary congenital glaucoma is effective in the reduction of the intraocular pressure.

RNA design rules from a massive open laboratory.

Self-assembling RNA molecules present compelling substrates for the rational interrogation and control of living systems. However, imperfect in silico models--even at the secondary structure level--hinder the design of new RNAs that function properly when synthesized. Here, we present a unique and potentially general approach to such empirical problems: the Massive Open Laboratory. The EteRNA project connects 37,000 enthusiasts to RNA design puzzles through an online interface. Uniquely, EteRNA participants not only manipulate simulated molecules but also control a remote experimental pipeline for high-throughput RNA synthesis and structure mapping. We show herein that the EteRNA community leveraged dozens of cycles of continuous wet laboratory feedback to learn strategies for solving in vitro RNA design problems on which automated methods fail. The top strategies--including several previously unrecognized negative design rules--were distilled by machine learning into an algorithm, EteRNABot. Over a rigorous 1-y testing phase, both the EteRNA community and EteRNABot significantly outperformed prior algorithms in a dozen RNA secondary structure design tests, including the creation of dendrimer-like structures and scaffolds for small molecule sensors. These results show that an online community can carry out large-scale experiments, hypothesis generation, and algorithm design to create practical advances in empirical science.

[Intraocular pressure in patients undergoing capsulotomy Nd: YAG laser].

OBJECTIVE: to demonstrate whether patients undergoing capsulotomy Nd: YAG laser developed intraocular hypertension after the procedure. METHODS: prospective, pre-experimental before and after 2 measurements with post-test in patients with posterior capsular opacity from the Ophthalmology Service. Measurements of intraocular pressure (IP) before capsulotomy Nd: YAG laser, and three hours and one week later. RESULTS: we studied 47 patients, 29.8 % were men and 70.2 % women. We compared between visual acuity before and one week later (Wilcoxon test p = 0.00). IP after three hours and one week later, comparisons with Friedman test were done (p =0. 002). We compared the IP prior and one week later (Wilcoxon test, with p =0.815). IP before and three hours later were obtained (p = 0.001) and IP three hours and one week later ( p = 0.004). CONCLUSIONS: we found an increase in IP in the first hours after the capsulotomy Nd: YAG laser, which decrease gradually until reaching the values presented before the procedure, in not more than a week period. It is a quick and safe procedure to treat posterior capsular opacity.

Lactation is a natural model of hippocampus neuroprotection against excitotoxicity.

Lactation is a temporary but complex physiological condition in which hormones and neurogenic stimulation from suckling cause maternal brain plasticity. It has been shown that lactation prevents cell damage induced by excitotoxicity in the dorsal hippocampus of the dam after peripheral administration of kainic acid (KA). The aim of this study was to determine whether lactation protects the maternal hippocampus against damage induced by intracerebral application (ICV) of KA and if lactation decreases, or only delays, this damaging effect of KA. Cell damage was assessed by Fluoro-Jade C staining in the hippocampus of virgin and lactating rats 24 or 72 h after ICV KA. Lactation prevented cell damage of the pyramidal layers of the hippocampus (CA1, CA3, and CA4), as compared to virgin rats. The longer period of KA exposure increased the difference in cell damage between these two conditions. The present results confirm that lactation is a natural model for neuroprotection, since it effectively prevents acute and chronic cell damage of the hippocampus induced by exposure to KA.