COLQ-Congenital myasthenic syndrome in an Iranian cohort: the clinical and genetics spectrum.

BACKGROUND: Congenital myasthenic syndrome (CMS) is a group of neuromuscular disorders caused by abnormal signal transmission at the motor endplate. Mutations in the collagen-like tail subunit gene (COLQ) of acetylcholinesterase are responsible for recessive forms of synaptic congenital myasthenic syndromes with end plate acetylcholinesterase deficiency. Clinical presentation includes ptosis, ophthalmoparesis, and progressive weakness with onset at birth or early infancy. METHODS: We followed 26 patients with COLQ-CMS over a mean period of 9 years (ranging from 3 to 213 months) and reported their clinical features, electrophysiologic findings, genetic characteristics, and therapeutic management. RESULTS: In our population, the onset of symptoms ranged from birth to 15 years. Delayed developmental motor milestones were detected in 13 patients (∼ 52%), and the most common presenting signs were ptosis, ophthalmoparesis, and limb weakness. Sluggish pupils were seen in 8 (∼ 30%) patients. All patients who underwent electrophysiologic study showed a significant decremental response (> 10%) following low-frequency repetitive nerve stimulation. Moreover, double compound muscle action potential was evident in 18 patients (∼ 75%). We detected 14 variants (eight novel variants), including six missense, three frameshift, three nonsense, one synonymous and one copy number variation (CNV), in the COLQ gene. There was no benefit from esterase inhibitor treatment, while treatment with ephedrine and salbutamol was objectively efficient in all cases. CONCLUSION: Despite the rarity of the disease, our findings provide valuable information for understanding the clinical and electrophysiological features as well as the genetic characterization and response to the treatment of COLQ-CMS.

MRI biomarkers and neuropsychological assessments of hippocampal and parahippocampal regions affected by ALS: A systematic review.

BACKGROUND AND OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a progressive motor and extra-motor neurodegenerative disease. This systematic review aimed to examine MRI biomarkers and neuropsychological assessments of the hippocampal and parahippocampal regions in patients with ALS. METHODS: A systematic review was conducted in the Scopus and PubMed databases for studies published between January 2000 and July 2023. The inclusion criteria were (1) MRI studies to assess hippocampal and parahippocampal regions in ALS patients, and (2) studies reporting neuropsychological data in patients with ALS. RESULTS: A total of 46 studies were included. Structural MRI revealed hippocampal atrophy, especially in ALS-FTD, involving specific subregions (CA1, dentate gyrus). Disease progression and genetic factors impacted atrophy patterns. Diffusion tensor imaging (DTI) showed increased mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), and decreased fractional anisotropy (FA) in the hippocampal tracts and adjacent regions, indicating loss of neuronal and white matter integrity. Functional MRI (fMRI) revealed reduced functional connectivity (FC) between the hippocampus, parahippocampus, and other regions, suggesting disrupted networks. Perfusion MRI showed hypoperfusion in parahippocampal gyri. Magnetic resonance spectroscopy (MRS) found changes in the hippocampus, indicating neuronal loss. Neuropsychological tests showed associations between poorer memory and hippocampal atrophy or connectivity changes. CA1-2, dentate gyrus, and fimbria atrophy were correlated with worse memory. CONCLUSIONS: The hippocampus and the connected regions are involved in ALS. Hippocampal atrophy disrupted connectivity and metabolite changes correlate with cognitive and functional decline. Specific subregions can be particularly affected. The hippocampus is a potential biomarker for disease monitoring and prognosis.

Clinical application of next generation sequencing for Mendelian disease diagnosis in the Iranian population.

Next-generation sequencing (NGS) has been proven to be one of the most powerful diagnostic tools for rare Mendelian disorders. Several studies on the clinical application of NGS in unselected cohorts of Middle Eastern patients have reported a high diagnostic yield of up to 48%, correlated with a high level of consanguinity in these populations. We evaluated the diagnostic utility of NGS-based testing across different clinical indications in 1436 patients from Iran, representing the first study of its kind in this highly consanguineous population. A total of 1075 exome sequencing and 361 targeted gene panel sequencing were performed over 8 years at a single clinical genetics laboratory, with the majority of cases tested as proband-only (91.6%). The overall diagnostic rate was 46.7%, ranging from 24% in patients with an abnormality of prenatal development to over 67% in patients with an abnormality of the skin. We identified 660 pathogenic or likely pathogenic variants, including 241 novel variants, associated with over 342 known genetic conditions. The highly consanguineous nature of this cohort led to the diagnosis of autosomal recessive disorders in the majority of patients (79.1%) and allowed us to determine the shared carrier status of couples for suspected recessive phenotypes in their deceased child(ren) when direct testing was not possible. We also highlight the observations of recessive inheritance of genes previously associated only with dominant disorders and provide an expanded genotype-phenotype spectrum for multiple less-characterized genes. We present the largest mutational spectrum of known Mendelian disease, including possible founder variants, throughout the Iranian population, which can serve as a unique resource for clinical genomic studies locally and beyond.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximises the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of novel symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryo-EM, we determine the structures of a precedented 60-mer icosahedral assembly and an unprecedented 36-mer tetrahedron that features significant geometric rearrangements around a novel interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple point mutation to various amino acids, and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent the first example of tetrahedral geometry across all characterised encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in protein sequence.

Neutral lipid storage disease with myopathy: clinicopathological and genetic features of nine Iranian patients.

The rare disorder known as Neutral Lipid Storage Disease with Myopathy presents with a variety of clinical manifestations, including myopathy, cardiac dysfunction, and other organ complications. Early diagnosis is crucial due to the increased risk of cardiomyopathy. We describe the clinical, histopathological, muscle imaging, and genetic findings of nine neutral lipid storage myopathy patients. Proximal weakness and asymmetric involvement may suggest lipid storage myopathy. While skeletal muscle weakness was the main manifestation in our patients, one case presented only with hyperCKemia. Additionally, three patients had fertility issues, two suffered from diabetes mellitus, two had cardiomyopathy, and one had a history of hypothyroidism. Muscle histopathology revealed lipid depositions and rimmed vacuoles, prompting peripheral blood smears to detect Jordan Anomalies. All muscle biopsies and peripheral blood smear showed lipid droplets, rimmed vacuoles, and Jordan anomaly. Identifying PNPLA2 gene mutations is important for diagnosing neutral lipid storage myopathy; our cases showed some novel mutations. This study highlights the importance of early diagnosis and comprehensive evaluation in managing neutral lipid storage myopathy cases.

Clinical and genetic features of patients suffering from CMT4J.

Mutations in the FIG4 gene have been identified in various diseases, including amyotrophic lateral sclerosis, Parkinson's disease, and Charcot-Marie-Tooth 4 J (CMT4J), with a wide range of phenotypic manifestations. We present eight cases of CMT4J patients carrying the p.Ile41Thr mutation of FIG4. The patients were categorized according to their phenotype. Six patients had a pure CMT; whereas, two patients had a CMT associated with parkinsonism. Three patients had an early onset and exhibited more severe forms of the disease. Three others experienced symptoms in their teenage years and had milder forms. Two patients had a late onset in adulthood. Four patients showed electrophysiological evidence of conduction blocks, typically associated with acquired neuropathies. Consequently, two of them received intravenous immunoglobulin treatment without a significant objective response. Interestingly, two heterozygous patients with the same mutations exhibited contrasting phenotypes, one having a severe early-onset form and the other experiencing a slow disease progression starting at the age of 49. Notably, although 7 out of 8 patients in this study were compound heterozygous for the p.Ile41Thr mutation, only one individual was found to be homozygous for this genetic variant and exhibited an early-onset, severe form of the disease. Additionally, one patient who developed the disease in his youth was also diagnosed with hereditary neuropathy with pressure palsies. Our findings provide insights into the CMT4J subtype by reporting on eight heterogeneous patient cases and highlight the potential for misdiagnosis when conduction blocks or asymmetrical nerve conduction study results are observed in patients with FIG4 mutations.

An uncommon presentation of early brainstem high-grade glioma in a 33-year-old male: A case study and review of literature.

INTRODUCTION AND IMPORTANCE: Unlike children, high-grade brainstem glioma (HG-BSG) in adults is a rare and diverse group of tumors. They can be classified based on their location and physical characteristics, which distinguishes them from pediatric brainstem gliomas. They are rare in adults, constituting only 1 % to 2 % of intracranial gliomas. They are often aggressive and have a poor prognosis, with a median survival time of 24 months. The diagnosis of brainstem gliomas typically involves a combination of clinical evaluation and imaging studies, mainly magnetic resonance imaging (MRI), which provides detailed images and can help identify the characteristics of the tumor. CASE PRESENTATION AND METHODS: We present a case study of an uncommon presentation of an early stage of HG-BSG in a 33-year-old male, who had a contrast-enhancing lesion in the ventrolateral medulla that extended to the lower aspect of the fourth ventricle and caused ventricular compression. CLINICAL DISCUSSION: The findings were consistent with the literature on the current state of HG-BSG MRI findings, which typically show contrast-enhancing, hyperintense, and infiltrative lesions that involve the pons, midbrain, or medulla oblongata. The diagnosis of HG-BSG was based on clinical and radiological criteria, as the patient refused to undergo a surgical biopsy. We also performed a literature review on the current state of brainstem HG-BSG MRI findings, summarizing the main features and patterns of these tumors. CONCLUSION: MRI can offer useful information regarding the tumor's location, size, and features, as well as its impact on surrounding tissues and cerebrospinal fluid circulation.

An interaction network approach predicts protein cage architectures in bionanotechnology.

Protein nanoparticles play pivotal roles in many areas of bionanotechnology, including drug delivery, vaccination, and diagnostics. These technologies require control over the distinct particle morphologies that protein nanocontainers can adopt during self-assembly from their constituent protein components. The geometric construction principle of virus-derived protein cages is by now fairly well understood by analogy to viral protein shells in terms of Caspar and Klug's quasi-equivalence principle. However, many artificial, or genetically modified, protein containers exhibit varying degrees of quasi-equivalence in the interactions between identical protein subunits. They can also contain a subset of protein subunits that do not participate in interactions with other assembly units, called capsomers, leading to gaps in the particle surface. We introduce a method that exploits information on the local interactions between the capsomers to infer the geometric construction principle of these nanoparticle architectures. The predictive power of this approach is demonstrated here for a prominent system in nanotechnology, the AaLS pentamer. Our method not only rationalises hitherto discovered cage structures but also predicts geometrically viable options that have not yet been observed. The classification of nanoparticle architecture based on the geometric properties of the interaction network closes a gap in our current understanding of protein container structure and can be widely applied in protein nanotechnology, paving the way to programmable control over particle polymorphism.

What is the Next Step after an Electrodiagnostic Study in Children with Polyneuropathies? Rationale for Laboratory and Other Diagnostic Tests.

The etiology of polyneuropathies varies in the pediatric population, where hereditary or metabolic disorders are far more common than in adults. However, treatable polyneuropathies, also prevalent in these settings, are those to prioritize. Moreover, diagnosing subacute and chronic symptoms in children can be challenging compared to adults. Therefore, selecting the best and most relevant laboratory investigations and paraclinical studies is critical. This taskcan be relatively challenging in countries with limited resources or insurance coverage. This study describe the various types of polyneuropathies found in children and their characteristics and suggest an algorithm for using the best laboratory tests in the context of the Iranian healthcare system.

Local structural flexibility drives oligomorphism in computationally designed protein assemblies.

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

Iron quantification in basal ganglia using quantitative susceptibility mapping in a patient with ALS: a case report and literature review.

Background: Quantitative susceptibility mapping (QSM) is a magnetic resonance imaging (MRI) technique that can measure the magnetic susceptibility of tissues, which can reflect their iron content. QSM has been used to detect iron accumulation in cortical and subcortical brain regions. However, its application in subcortical regions such as the basal ganglia, particularly the putamen, is rare in patients with amyotrophic lateral sclerosis (ALS). Case presentation and literature review: We present the case of a 40-year-old male patient with ALS who underwent an MRI for QSM. We compared his QSM images with those of a control subject and performed a quantitative analysis of the magnetic susceptibility values in the putamen regions. We also reviewed the literature on previous QSM studies in ALS and summarized their methods and findings. Our QSM analysis revealed increased magnetic susceptibility values in the bilateral putamen of the ALS patient compared to controls, indicating iron overload. This finding is consistent with previous studies reporting iron dysregulation in subcortical nuclei in ALS. We also discussed the QSM processing techniques used in our study and in the literature, highlighting their advantages and limitations. Conclusion: This case report demonstrates the potential of QSM as a sensitive MRI biomarker for evaluating iron levels in subcortical regions of ALS patients. QSM can provide quantitative information on iron deposition patterns in both motor and extra-motor areas of ALS patients, which may help understand the pathophysiology of ALS and monitor disease progression. Further studies with larger samples are needed to validate these results and explore the clinical implications of QSM in ALS.

MRI biomarkers for memory-related impairment in amyotrophic lateral sclerosis: a systematic review.

Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with cognitive and behavioral impairments and motor symptoms. Magnetic resonance imaging (MRI) biomarkers have been investigated as potential tools for detecting and monitoring memory-related impairment in ALS. Our objective was to examine the importance of identifying MRI biomarkers for memory-related impairment in ALS, motor neuron disease (MND), and ALS frontotemporal dementia (FTD) (ALS-FTD) patients. Methods: PubMed and Scopus databases were searched. Keywords covering magnetic resonance imaging, ALS, MND, and memory impairments were searched. There were a total of 25 studies included in our work here. Results: The structural MRI (sMRI) studies reported gray matter (GM) atrophy in the regions associated with memory processing, such as the hippocampus and parahippocampal gyrus (PhG), in ALS patients. The diffusion tensor imaging (DTI) studies showed white matter (WM) alterations in the corticospinal tract (CST) and other tracts that are related to motor and extra-motor functions, and these alterations were associated with memory and executive function impairments in ALS. The functional MRI (fMRI) studies also demonstrated an altered activation in the prefrontal cortex, limbic system, and other brain regions involved in memory and emotional processing in ALS patients. Conclusion: MRI biomarkers show promise in uncovering the neural mechanisms of memory-related impairment in ALS. Nonetheless, addressing challenges such as sample sizes, imaging protocols, and longitudinal studies is crucial for future research. Ultimately, MRI biomarkers have the potential to be a tool for detecting and monitoring memory-related impairments in ALS.

Iranian clinical practice guideline for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegeneration involving motor neurons. The 3-5 years that patients have to live is marked by day-to-day loss of motor and sometimes cognitive abilities. Enormous amounts of healthcare services and resources are necessary to support patients and their caregivers during this relatively short but burdensome journey. Organization and management of these resources need to best meet patients' expectations and health system efficiency mandates. This can only occur in the setting of multidisciplinary ALS clinics which are known as the gold standard of ALS care worldwide. To introduce this standard to the care of Iranian ALS patients, which is an inevitable quality milestone, a national ALS clinical practice guideline is the necessary first step. The National ALS guideline will serve as the knowledge base for the development of local clinical pathways to guide patient journeys in multidisciplinary ALS clinics. To this end, we gathered a team of national neuromuscular experts as well as experts in related specialties necessary for delivering multidisciplinary care to ALS patients to develop the Iranian ALS clinical practice guideline. Clinical questions were prepared in the Patient, Intervention, Comparison, and Outcome (PICO) format to serve as a guide for the literature search. Considering the lack of adequate national/local studies at this time, a consensus-based approach was taken to evaluate the quality of the retrieved evidence and summarize recommendations.

The First Report of Iranian Registry of Patients with Spinal Muscular Atrophy.

BACKGROUND: Insufficient amounts of survival motor neuron protein is leading to one of the most disabling neuromuscular diseases, spinal muscular atrophy (SMA). Before the current study, the detailed characteristics of Iranian patients with SMA had not been determined. OBJECTIVE: To describe the key demographic, clinical, and genetic characteristics of patients with SMA registered in the Iranian Registry of SMA (IRSMA). METHODS: IRSMA has been established since 2018, and the demographic, clinical, and genetic characteristics of patients with SMA were recorded according to the methods of treat neuromuscular disease (TREAT-NMD) project. RESULTS: By October 1, 2022, 781 patients with 5q SMA were registered. Of them, 164 patients died, the majority of them had SMA type 1 and died during the first 20 months of life. The median survival of patients with type 1 SMA was 23 months. The consanguinity rate in 617 alive patients was 52.4%, while merely 24.8% of them had a positive family history. The most common type of SMA in live patients was type 3. Morbidities were defined as having scoliosis (44.1%), wheelchair dependency (36.8%), tube feeding (8.1%), and requiring mechanical ventilation (9.9%). Most of the registered patients had a homozygous deletion of SMN1, while the frequency of patients with higher copy numbers of SMN2, was less in more severe types of the disease. Earlier onset of the disease was significantly seen in patients with lower copy numbers of SMN2. The neuronal apoptosis inhibitory protein (NAIP) gene deletion was associated with a higher incidence of more severe types of SMA, higher dependency on ventilators, tube feeding, and earlier onset of the disease. CONCLUSIONS: The IRSMA is the first established Iranian nationwide registry of patients with SMA. Using this registry, decision-makers, researchers, and practitioners can precisely understand the epidemiology, characteristics, and genetics of patients with SMA in Iran.

Comparison of Clinical, Ultrasound, and Electrophysiologic Changes in Chronic Inflammatory Demyelinating Polyneuropathy: A Prospective Study.

PURPOSE: The follow-up and monitoring of response to immunomodulatory therapy in patients with chronic inflammatory demyelinating polyneuropathy are still challenging. Various outcome measures have been proposed in recent years, and some are now frequently used in daily clinical practice; however, reliable biomarkers for the disease activity and treatment response are lacking. METHODS: Cross-sectional nerve area of the bilateral vagus, fifth and the sixth cervical spinal, median, ulnar, tibial, peroneal, and sural nerves were measured at 2 time points with an interval of 6 months using nerve ultrasound. The results were used to calculate the ultrasound pattern sumscore (UPSS). The correlation between UPSS change (ΔUPSS) and changes in functional and nerve conduction studies measures over the study period were assessed. RESULTS: Sixteen patients completed this prospective, observational study. General linear model showed that ΔUPSS is significantly associated with ΔMedical Research Council sumscore (ß = -0.72, P = 0.003), Δhandgrip strength (ß = -0.57, P = 0.014), ΔRasch-built overall disability scale (ß = -0.57, P = 0.010), and Δoverall neuropathy limitations scale (ß = 0.75, P < 0.001), after adjustment of confounding variables. Nevertheless, ΔUPSS was not correlated with other clinical measures, including Δpinch power, Δ9-hole peg test, Δ10-m walking test, and Δnerve conduction study sumscore ( P values > 0.05). CONCLUSIONS: Nerve ultrasound might be an efficient method for monitoring the functional status of patients with chronic inflammatory demyelinating polyneuropathy over time because the alterations in its scores could significantly reflect clinical changes.

Investigating the Association Between Muscular Ultrasonographic Alterations and Clinical Symptoms in Patients With Inflammatory Myopathy.

Introduction: Muscle biopsy is commonly used to diagnose inflammatory myopathies. We evaluated the ability of muscle ultrasound, a non-invasive and simple tool, to distinguish between healthy subjects and patients with inflammatory myopathy. Methods: This study was conducted on 17 patients recently diagnosed with biopsy inflammatory myopathies (12 dermatomyositis, 5 polymyositis) compared with 17 age- and gender-matched healthy control adults. All patients underwent clinical assessments, including manual muscle testing, hand-held dynamometry, and muscle ultrasound evaluations, including thickness and echo intensity in predefined muscle groups. Results: The disease duration was seven months (interquartile range: 3 to 11 months). Except for the biceps and gastrocnemius, patients' muscles had significantly higher echo intensity and lower thickness than the control group. The echo intensity sum-score manifested the highest area under the curve compared to the sum-scores of other variables (echo intensity vs manual muscle testing: Area under curves-difference=0.18, P<0.01; echo intensity vs dynamometry: Area under curves-difference=0.14, P=0.02; echo intensity vs thickness: Area under curves-differences-difference=0.25, P<0.01). Conclusion: The echo intensity of muscles differed significantly between healthy individuals and patients with inflammatory myopathies and may serve as a useful diagnostic biomarker.

Clinical features and outcomes of patients with myasthenia gravis affected by COVID-19: A single-center study.

INTRODUCTION: Myasthenia Gravis (MG) is an autoimmune disorder that can exacerbate for various reasons including infections. In this study, we describe clinical symptoms, outcomes, and management of MG patients affected by COVID-19 infection. METHODS: This observational retrospective study was performed on patients previously diagnosed as MG, presenting with COVID-19 in the clinic or emergency department between April 2020 and August 2021. The clinical data, outcome, and therapeutic interventions were assessed in 83 patients with MG and COVID-19 infection. RESULTS: Seventy-seven patients performed PCR testing for COVID-19, of which 73 (94.8 %) were positive. Seven patients had the positive serologic test for COVID-19 (IgG and IgM). Fifty-seven (68.7 %) patients had lung involvement. Thirty-five (42.1 %) of patients were admitted to the hospital. Twelve (14.5 %) patients needed hospitalization in an intensive care unit (ICU), with a mean stay of 7.36 ± 5.6 days (rang: 2-20 days). Four (4.8 %) patients were intubated and required mechanical ventilation. Sixteen (19.3 %) patients experienced an exacerbation of myasthenia gravis and were treated with PLEX (n = 2), IVIG (n = 7), and intravenous (IV) methylprednisolone (n = 7). The outcome was favorable in 79 patients and fatal in four patients, three of whom had other comorbidities. One patient died due to severe COVID-19 involvement. CONCLUSION: The findings from our study demonstrated that patients with previous MG concurrence with COVID-19 have favorable clinical outcomes. Most patients did not need to be hospitalized and more than 80 % of patients did not display MG exacerbation.

Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation.

Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro, which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5' labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.