SimplyFire: An Open-Source, Customizable Software Application for the Analysis of Synaptic Events.

We have developed an open-source software for neuroscientists to analyze electrophysiological recordings. Named SimplyFire, the software gives the users the flexibility to analyze a variety of recordings using an interactive graphical user interface or as an importable Python package. The software features a simple plugin structure that allows users to create and deploy various electrophysiology analysis tools. SimplyFire is pre-packaged with tools commonly used in electrophysiology, such as noise filtering, trace averaging, miniature analysis, and trace exporting. We discuss in detail the algorithm behind the different features of the analysis tool. We verify the accuracy of the algorithm by testing the software using computer-generated traces with known true values of the events. SimplyFire will be distributed under the GPLv3.0 license. The open nature of this software will allow interested investigators to modify and expand the software for additional capabilities as needed. We believe this software will not only compete with commercially available software packages but will also present a powerful tool to meet the current and unmet needs of electrophysiologists.

N6-Adenosine Methylation of SARS-CoV-2 5'-UTR Regulates Translation.

The coronavirus disease 2019 (COVID19) continues to spread despite global vaccination efforts (1). This, alongside the rapid emergence of vaccine resistant variants, creates a need for orthogonal therapeutic strategies targeting more conserved facets of severe acute respiratory syndrome coronavirus (SARS-CoV-2) (2-4). One conserved feature of all coronaviruses is their ability to undergo discontinuous transcription wherein individual open reading frames fuse with the 5'-UTR leader sequence during negative-strand RNA synthesis (5). As such all viral protein coding genes use the same 5'-UTR for translation (6). Using in vitro reporter assays, we demonstrate that the SARS-CoV-2 5'-UTR efficiently initiates protein translation despite its predicted structural complexity. Through a combination of bioinformatic and biochemical assays, we demonstrate that a single METTL3-dependent m6A methylation event in SARS-CoV-2 5'-UTR regulates the rate of translation initiation. We show that m6A likely exerts this effect by destabilizing secondary structure in the 5'-UTR, thereby facilitating access to the ribosomal pre-initiation complex. This discovery opens new avenues for novel therapeutic strategies aimed at controlling the ability of SARS-CoV-2 to replicate in host cells.

Delta/Notch signaling in glia maintains motor nerve barrier function and synaptic transmission by controlling matrix metalloproteinase expression.

While the role of barrier function in establishing a protective, nutrient-rich, and ionically balanced environment for neurons has been appreciated for some time, little is known about how signaling cues originating in barrier-forming cells participate in maintaining barrier function and influence synaptic activity. We have identified Delta/Notch signaling in subperineurial glia (SPG), a crucial glial type for Drosophila motor axon ensheathment and the blood-brain barrier, to be essential for controlling the expression of matrix metalloproteinase 1 (Mmp1), a major regulator of the extracellular matrix (ECM). Our genetic analysis indicates that Delta/Notch signaling in SPG exerts an inhibitory control on Mmp1 expression. In the absence of this inhibition, abnormally enhanced Mmp1 activity disrupts septate junctions and glial ensheathment of peripheral motor nerves, compromising neurotransmitter release at the neuromuscular junction (NMJ). Temporally controlled and cell type-specific transgenic analysis shows that Delta/Notch signaling inhibits transcription of Mmp1 by inhibiting c-Jun N-terminal kinase (JNK) signaling in SPG. Our results provide a mechanistic insight into the regulation of neuronal health and function via glial-initiated signaling and open a framework for understanding the complex relationship between ECM regulation and the maintenance of barrier function.

Dichotomous cis-regulatory motifs mediate the maturation of the neuromuscular junction by retrograde BMP signaling.

Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-ß-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. Here, we uncover a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. Our findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling.

Loss of 4E-BP converts cerebellar long-term depression to long-term potentiation.

Genetic perturbances in translational regulation result in defects in cerebellar motor learning; however, little is known about the role of translational mechanisms in the regulation of cerebellar plasticity. We show that genetic removal of 4E-BP, a translational suppressor and target of mammalian target of rapamycin complex 1, results in a striking change in cerebellar synaptic plasticity. We find that cerebellar long-term depression (LTD) at parallel fiber-Purkinje cell synapses is converted to long-term potentiation in 4E-BP knockout mice. Biochemical and pharmacological experiments suggest that increased phosphatase activity largely accounts for the defects in LTD. Our results point to a model in which translational regulation through the action of 4E-BP plays a critical role in establishing the appropriate kinase/phosphatase balance required for normal synaptic plasticity in the cerebellum.

A Neuron-Glial Trans-Signaling Cascade Mediates LRRK2-Induced Neurodegeneration.

Pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) induce an age-dependent loss of dopaminergic (DA) neurons. We have identified Furin 1, a pro-protein convertase, as a translational target of LRRK2 in DA neurons. Transgenic knockdown of Furin1 or its substrate the bone morphogenic protein (BMP) ligand glass bottom boat (Gbb) protects against LRRK2-induced loss of DA neurons. LRRK2 enhances the accumulation of phosphorylated Mad (pMad) in the nuclei of glial cells in the vicinity of DA neurons but not in DA neurons. Consistently, exposure to paraquat enhances Furin 1 levels in DA neurons and induces BMP signaling in glia. In support of a neuron-glial signaling model, knocking down BMP pathway members only in glia, but not in neurons, can protect against paraquat toxicity. We propose that a neuron-glial BMP-signaling cascade is critical for mediating age-dependent neurodegeneration in two models of Parkinson's disease, thus opening avenues for future therapeutic interventions.

Removing 4E-BP Enables Synapses to Refine without Postsynaptic Activity.

Throughout the developing nervous system, considerable synaptic re-organization takes place as postsynaptic neurons extend dendrites and incoming axons refine their synapses, strengthening some and eliminating others. It is well accepted that these processes rely on synaptic activity; however, the mechanisms that lead to this developmental reorganization are not fully understood. Here, we explore the regulation of cap-dependent translation, a mechanism known to play a role in synaptic growth and plasticity. Using sympathetic ganglia in α3 nicotinic acetylcholine receptor (nAChR)-knockout (KO) mice, we establish that electrophysiologically silent synapses between preganglionic axons and postsynaptic sympathetic neurons do not refine, and the growth of dendrites and the targeting of synapses on postsynaptic neurons are impaired. Remarkably, genetically removing 4E-BP, a suppressor of cap-dependent translation, from these α3 nAChR-KO mice largely restores these features. We conclude that synaptic connections can re-organize and refine without postsynaptic activity during post-natal development when 4E-BP-regulated cap-dependent translation is enhanced.

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction.

Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.

Post-transplantation Presentation of ANCA-associated Vasculitis: Granulomatosis with Polyangitis.

Granulomatosis with polyangitis (GPA) is characterized by necrotizing granulomatosis of the upper and lower respiratory tract and glomerulonephritis. If GPA does not respond to appropriate management, it might result in end-stage renal disease, which may remit the disease severity. The overall impression is that immunosuppression following renal transplantation would further subside the vasculitis. However, several studies have shown that systemic vasculitis recur in 25% of patients following renal transplantation. This may indicate the perplexing nature of the immune system. One of the key factors in prevention of relapse of GPA is following up of patients by careful immunosuppressive dose adjustment and regular measurement of biomarkers for vasculitis. Herein, we describe an interesting case of biopsy-proven GPA who had a complex long history of several post-transplantation relapses in different organs with anti-neutrophil cytoplasmic antibodies seroconversion. This case emphasizes that vasculitis in particular GPA can mimic various diseases depending on which vessels and organs are affected by the inflammation and is one of the reversible causes of failure of transplanted kidney. Bearing the diagnosis in mind as one of the potential differential diagnoses of failure of renal transplantation will lead to early diagnosis and treatment of recurrent GPA.

[Mitochondrial DNA deletion Δ4977 in peptic ulcer disease].

Reactive oxygen species (ROS) play a critical role in peptic ulcer disease (PUD). Due to the high rate of ROS production and limited capacity for DNA repair within mitochondria, mtDNA is susceptible to oxidative damage and mutations. mtDNA deletion Δ4977 is one of the most common deletion events identified in mitochondria. We examined the association of 4977-bp mtDNA deletion with PUD. Genotypes were determined in bioptic samples of 150 PUD patients and 190 controls. The 4977-bp mtDNA deletion was found more frequently among patients with PUD (52%) than among controls (22.63%). The strong association between the mtDNA 4977-bp deletion and PUD was confirmed (OR = 3.7; 95% CI, 2.32-5.91; P = 0.0001). The 4977-bp deletion in mitochondrial DNA may be a risk factor for PUD, or may reflect an increase in oxidative stress that commonly accompanies underlying PUD disease. Larger population-based studies are needed to uncover the possible causal relationship between this deletion and peptic ulcer disease.

PCR-RFLP analysis of Plasmodium vivax reticulocyte binding protein2c gene in field isolates of Iran.

A family of reticulocyte-binding proteins of Plasmodium vivax (PvRBP) is localised at the apical pole of the merozoites and appears to bind to reticulocytes specifically and has also been involved in identifying host cells. Protein component produced by the Pvrbp2c gene is highly antigenic. The aim of this study was to detect the genetic diversity in the Pvrbp2c gene of Iranian P. vivax field isolates using the polymerase chain reaction- restricted fragment length polymorphism (PCR-RFLP) technique. A total of 79 P. vivax malaria patients with fever participated in the study. Alu1 and Apo1 restriction enzymes were independently used to identify allelic variants of the Pvrbp2c gene. All of the samples exhibited a single band of about 2 Kb in nested PCR. Among 79 P. vivax field isolates in the RFLP with Apo1 and Alu1 restriction enzymes, 15 and nine patterns were observed, respectively. In total, 24 various patterns were detected from the combined findings of both Alu1 and Apo1 fragments in RFLP. This study revealed that Pvrbp2c has genetic diversity in southeast Iran. Genotyping of Pvrbp2c not only shows the heterogeneity of P. vivax but also provides important information that could be used to control vivax malaria.

The amoebicidal activity of Ziziphus vulgaris extract and its fractions on pathogenic Acanthamoeba trophozoites and cysts.

Acanthamoeba genus includes pathogenic species which are causal agents of a severe sight-threatening infection of the eye known as Acanthamoeba keratitis (AK). Furthermore, the number of AK is worryingly increasing worldwide, mostly in contact lens users. Until present, there is a general failure to reach a fully effective treatment against AK which is mainly due to the amoebic double-walled cyst stage which forms a protective barrier against drugs. Therefore, drug discovery research towards AK treatment is a must. In this study, Ziziphus vulgaris, a native plant of Asian countries, was checked for its activity against Acanthamoeba. For this purpose and in order to determine the in vitro amoebicidal effects of Ziziphus vulgaris aqueous extract and its fractions (chloroformic, remaining aqueous and primary alcoholic) against Acanthmoeba trophozoites and cysts, activity and sensitivity assays were performed. Moreover, the toxic effect of the extract and its fractions was also tested on murine peritoneal macrophages using a colorimetric tetrazolium salt (MTT) test. The obtained results showed that the chloroformic fraction presented a higher anti-Acanthamoeba activity when compared to the other fractions (Trophozoites/cysts were eliminated, when incubated in a concentration of 50 mg/ml of the fraction, after 24 hours). The calculated active concentrations against Acanthamoeba of these extracts did not shown any high cytotoxicity levels.This study suggests that the Ziziphus vulgaris chloroformic fraction, may present compounds with relevance for the development of novel antiAcanthamoeba drugs.

PCR-RFLP analysis of Plasmodium vivax reticulocyte binding protein2c gene in field isolates of Iran

A family of reticulocyte-binding proteins of Plasmodium vivax (PvRBP) is localised at the apical pole of the merozoites and appears to bind to reticulocytes specifically and has also been involved in identifying host cells. Protein component produced by the Pvrbp2c gene is highly antigenic. The aim of this study was to detect the genetic diversity in the Pvrbp2c gene of Iranian P. vivax field isolates using the polymerase chain reaction- restricted fragment length polymorphism (PCR-RFLP) technique. A total of 79 P. vivax malaria patients with fever participated in the study. Alu1 and Apo1 restriction enzymes were independently used to identify allelic variants of the Pvrbp2c gene. All of the samples exhibited a single band of about 2 Kb in nested PCR. Among 79 P. vivax field isolates in the RFLP with Apo1 and Alu1 restriction enzymes, 15 and nine patterns were observed, respectively. In total, 24 various patterns were detected from the combined findings of both Alu1 and Apo1 fragments in RFLP. This study revealed that Pvrbp2c has genetic diversity in southeast Iran. Genotyping of Pvrbp2c not only shows the heterogeneity of P. vivax but also provides important information that could be used to control vivax malaria.

Acute Fasting Regulates Retrograde Synaptic Enhancement through a 4E-BP-Dependent Mechanism.

While beneficial effects of fasting on organismal function and health are well appreciated, we know little about the molecular details of how fasting influences synaptic function and plasticity. Our genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, our findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. Our results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits.

Evaluation of modified Ziehl-Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens.

To determine the sensitivity and specificity of routine screening methods for cryptosporidiosis, three methods including conventional modified Ziehl-Neelsen (MZN), direct fluorescent-antibody (DFA) and Nested-PCR assay compared together. To this end, their ability to identify the low concentrations of Cryptosporidium spp. oocysts in children fecal samples was evaluated. The sample population of this study was children under 12 years old who had diarrhea and referred to pediatric hospitals in Tehran, Iran. 2,510 stool specimens from patients with diarrhea were screened for Cryptosporidium oocysts by concentration method and MZN. To determine sensitivity and specificity, Nested-PCR and DFA were performed on 30 positive and 114 negative samples which previously had been proved by MZN. By using the microscopic method, DFA assay and PCR analysis, a total of 30 (1.2 %), 28 (1.1 %) and 32 (1.27 %) positive samples were detected respectively. According to the results, the sensitivity, specificity, and positive and negative predictive values of the Nested-PCR assay were 100 %, compared to 94, 100, 100, and 98 %, respectively, for MZN and 87.5, 100, 100, and 96 %, respectively, for DFA. Results of the present study showed that the Nested-PCR assay was more sensitive than the other two methods and laboratories can use the Nested-PCR method for precise diagnosis of Cryptosporidium spp. However, regarding the costs of Nested-PCR and its unavailability in all laboratories and hospitals, MZN staining on smears has also enough accuracy for Cryptosporidium diagnosis.

LRRK2 regulates retrograde synaptic compensation at the Drosophila neuromuscular junction.

Parkinson's disease gene leucine-rich repeat kinase 2 (LRRK2) has been implicated in a number of processes including the regulation of mitochondrial function, autophagy and endocytic dynamics; nevertheless, we know little about its potential role in the regulation of synaptic plasticity. Here we demonstrate that postsynaptic knockdown of the fly homologue of LRRK2 thwarts retrograde, homeostatic synaptic compensation at the larval neuromuscular junction. Conversely, postsynaptic overexpression of either the fly or human LRRK2 transgene induces a retrograde enhancement of presynaptic neurotransmitter release by increasing the size of the release ready pool of vesicles. We show that LRRK2 promotes cap-dependent translation and identify Furin 1 as its translational target, which is required for the synaptic function of LRRK2. As the regulation of synaptic homeostasis plays a fundamental role in ensuring normal and stable synaptic function, our findings suggest that aberrant function of LRRK2 may lead to destabilization of neural circuits.

Presence of Balamuthia mandrillaris in hot springs from Mazandaran province, northern Iran.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause cutaneous lesions and Balamuthia amoebic encephalitis. The biology and environmental distribution of B. mandrillaris is still poorly understood and isolation of this pathogen from the environment is a rare event. Previous studies have reported that the presence of B. mandrillaris in the environment in Iran may be common. However, no clinical cases have been reported so far in this country. In the present study, a survey was conducted in order to evaluate the presence of B. mandrillaris in hot-spring samples of northern Iran. A total of 66 water samples were analysed using morphological and molecular tools. Positive samples by microscopy were confirmed by performing PCR amplification of the 16S rRNA gene of B. mandrillaris. Sequencing of the positive amplicons was also performed to confirm morphological data. Two of the 66 collected water samples were positive for B. mandrillaris after morphological and molecular identification. Interestingly, both positive hot springs had low pH values and temperatures ranging from 32 °C to 42 °C. Many locals and tourists use both hot springs due to their medicinal properties and thus contact with water bodies containing the organism increases the likelihood of infection. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from hot-spring sources related to human activity. Therefore, B. mandrillaris should be considered as a possible causative agent if cases of encephalitis are suspected following immersion in hot springs in addition to Acanthamoeba and Naegleria.

Glanzmann thrombasthenia in Pakistan: molecular analysis and identification of novel mutations.

Glanzmann thrombasthenia (GT) is an inherited genetic disorder affecting platelets, which is characterized by spontaneous mucocutaneous bleeding and abnormally prolonged bleeding in response to injury or trauma. The underlying defect is failure of platelet aggregation due to qualitative and/or quantitative deficiency of platelet integrin αIIbß3 resulting from molecular genetic defects in either ITGA2B or ITGB3. Here, we examine a Pakistani cohort of 15 patients with clinical symptoms of GT who underwent laboratory and molecular genetic analysis. In patients with a broad range of disease severity and age of presentation, we identified pathogenic mutations in ITGA2B in 11 patients from 8 different families, including 2 novel homozygous mutations and 1 novel heterozygous mutation. Mutations in ITGB3 were identified in 4 patients from 3 families, two of which were novel homozygous truncating mutations. A molecular genetic diagnosis was established in 11 families with GT, including 5 novel mutations extending the spectrum of mutations in this disease within a region of the world where little is known about the incidence of GT. Mutational analysis is a key component of a complete diagnosis of GT and allows appropriate management and screening of other family members to be performed.

Congenital generalized lipodystrophy: identification of novel variants and expansion of clinical spectrum.

Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder with two major subtypes. Variants in AGPAT2 result in CGL type 1 with milder manifestations, whereas BSCL2 variants cause CGL type 2 with more severe features. Muscle hypertrophy caused by lack of adipose tissue is present early in life in CGL patients. Our aim was to investigate 10 CGL patients from 7 different countries and report genotype-phenotype relationships. Genetic analysis identified disease-causing variants in AGPAT2 (five patients) and in BSCL2 (five patients), including three novel variants; c.134C>A (p.Ser45*), c.216C>G (p.Tyr72*) in AGPAT2 and c.458C>A (p.Ser153*) in BSCL2. We also report possible novel clinical features such as anemia, breast enlargement, steatorrhea, intraventricular hemorrhage and nephrolithiasis in CGL patients. Generalized lipodystrophy and muscular hypertrophy were the only features in all of our patients. Hepatomegaly was the second common feature. Some manifestations were exclusively noticed in our CGL2 patients; hypertrichosis, high-pitched voice and umbilical hernia. Bone cysts and history of seizures were noticed only in CGL1 patients. The findings of this study expand our knowledge of genotype-phenotype correlations in CGL patients. These results have important clinical applications in diagnosis and management of the CGL patients as well as in genetic counseling in families at-risk.

Identification of an NPHP1 deletion causing adult form of nephronophthisis.

AIMS: Nephronophthisis (NPHP) is an autosomal recessive cystic disease of the kidney with main characteristic features of polyuria/polydipsia, mild or absent proteinuria, interstitial fibrosis, and tubular cysts. NPHP is responsible for 5-10 % of inheritable end-stage renal disease (ESRD) cases. We investigated the clinical features and genetic cause of NPHP in a Persian family with three siblings affected by tubulointerstitial nephropathy reaching ESRD in adulthood. METHODS: Uromodulin (UMOD), known to be involved in adult medullary cystic kidney disease, and nephronophthisis 1 (NPHP1) were investigated in the genomic DNA of the probands using DNA sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis and molecular karyotyping. RESULTS: No mutation was detected in UMOD. Copy number variation analysis of the NPHP1 gene using the commercially available MLPA kit identified a recurrent large homozygous deletion encompassing all NPHP1 exons. The parents were heterozygous for this deletion. Whole genome array-CGH analysis confirmed a homozygous deletion on chromosome 2q13, NPHP1 site, and revealed that the size of the copy number loss was approximately 102 Kbp. CONCLUSION: This is the first report of determination of an NPHP1 deletion size using routine diagnostic methods. The results of this study expand the knowledge about the genotype-phenotype correlations in NPHP1, and have implications for genetic counseling and family planning advice for other affected families. This is the first molecular analysis of NPHP1 in an Iranian kindred.