Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration.

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

Inhibition of NF-κB-Mediated Proinflammatory Transcription by Ru(II) Complexes of Anti-Angiogenic Ligands in Triple-Negative Breast Cancer.

Nuclear factor kappa beta (NF-κB) plays a pivotal role in breast cancer, particularly triple-negative breast cancer, by promoting inflammation, proliferation, epithelial-mesenchymal transition, metastasis, and drug resistance. Upregulation of NF-κB boosts vascular endothelial growth factor (VEGF) expression, assisting angiogenesis. The Ru(II) complexes of methyl- and dimethylpyrazolyl-benzimidazole N,N donors inhibit phosphorylation of ser536 in p65 and translocation of the NF-κB heterodimer (p50/p65) to the nucleus, disabling transcription to upregulate inflammatory signaling. The methyl- and dimethylpyrazolyl-benzimidazole inhibit VEGFR2 phosphorylation at Y1175, disrupting downstream signaling through PLC-γ and ERK1/2, ultimately suppressing Ca(II)-signaling. Partial release of the antiangiogenic ligand in a reactive oxygen species-rich environment is possible as per our observation to inhibit both NF-κB and VEGFR2 by the complexes. The complexes are nontoxic to zebrafish embryos up to 50 µM, but the ligands show strong in vivo antiangiogenic activity at 3 µM during embryonic growth in Tg(fli1:GFP) zebrafish but no visible effect on the adult phase.

A Case of Primary Amebic Meningoencephalitis Associated with Surfing at an Artificial Surf Venue: Environmental Investigation.

Naegleria fowleri is a thermophilic ameba found in freshwater that causes primary amebic meningoencephalitis (PAM) when it enters the nose and migrates to the brain. In September 2018, a 29-year-old man died of PAM after traveling to Texas. We conducted an epidemiologic and environmental investigation to identify the water exposure associated with this PAM case. The patient's most probable water exposure occurred while surfing in an artificial surf venue. The surf venue water was not filtered or recirculated; water disinfection and water quality testing were not documented. N. fowleri and thermophilic amebae were detected in recreational water and sediment samples throughout the facility. Codes and standards for treated recreational water venues open to the public could be developed to address these novel venues. Clinicians and public health officials should also consider novel recreational water venues as a potential exposure for this rare amebic infection.

NOTCH pathway inactivation reprograms stem-like oral cancer cells to JAK-STAT dependent state and provides the opportunity of synthetic lethality.

BACKGROUND: We have recently provided the evidence of interconvertible cellular states, driving non-genetic heterogeneity among stem-like oral cancer cells (oral-SLCCs). Here, NOTCH pathway-activity status is explored as one of the possible mechanisms behind this stochastic plasticity. METHODS: Oral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivo effects were studied by xenograft growth in zebrafish embryo. RESULTS: We have observed stochastic plasticity in oral-SLCCs, spontaneously maintaining both NOTCH-active and inactive states. While cisplatin refraction was associated with post-treatment adaptation to the active-state of NOTCH pathway, oral-SLCCs with inactive NOTCH pathway status showed aggressive tumor growth and poor prognosis. RNAseq analysis clearly suggested the upregulation of JAK-STAT pathway in NOTCH pathway-inactive subset. The 3D-spheroids with lower NOTCH-activity status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib or siRNA mediated downregulation of tested partners STAT3/4. Oral-SLCCs were programmed to adapt the inactive status of NOTCH pathway by exposing to Î³-secretase inhibitors, LY411575 or RO4929097, followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This approach resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft initiation in Zebrafish embryos. CONCLUSION: Study revealed for the first time that NOTCH pathway-inactive state exhibit activation of JAK-STAT pathways, as synthetic lethal pair. Therefore, co-inhibition of these pathway may serve as novel therapeutic strategy against aggressive oral cancer.

The Epidemiology and Clinical Features of Non-Keratitis Acanthamoeba Infections in the United States, 1956-2020.

Background: Acanthamoeba is a free-living ameba that can cause severe disease affecting the central nervous system, skin, sinuses, and other organs, particularly in immunocompromised individuals. These rare but severe infections are often fatal, yet incompletely described. Methods: Cases included were either reported to the Centers for Disease Control and Prevention (CDC) Free-Living Ameba program or published in scientific literature. Characteristics of all patients in the United States with laboratory-confirmed non-keratitis Acanthamoeba infections were described using descriptive statistics, and associations with survival were determined using χ2 and Fisher exact tests. Results: Of 173 patients identified, 71% were male and the median age was 44 years (range, 0-87 years). Of these, 26 (15%) survived. Most patients (88%) had at least 1 immunocompromising condition, most commonly human immunodeficiency virus (39%), cancer (28%), and solid organ or hematopoietic stem cell transplant (28%). Granulomatous amebic encephalitis (GAE) was the most common disease presentation (71%). Skin (46%), sinuses (29%), lungs (13%), and bone (6%) were also involved. Nearly half of patients (47%) had involvement of >1 organ system. Survival was less frequent among those with GAE (3%, P < .001) compared with cutaneous disease, rhinosinusitis, or multiorgan disease not including GAE. Of 7 who received the currently recommended treatment regimen, 5 (71%) survived. Conclusions: Non-keratitis Acanthamoeba infections occur primarily in immunocompromised individuals and are usually fatal. Survival may be associated with disease presentation and treatment. Providers who care for at-risk patients should be aware of the various disease manifestations to improve early recognition and treatment.

Cytotoxic Ruthenium(II) Complexes of Pyrazolylbenzimidazole Ligands That Inhibit VEGFR2 Phosphorylation.

Eight new ruthenium(II) complexes of N,N-chelating pyrazolylbenzimidazole ligands of the general formula [RuII(p-cym)(L)X]+ [where the ligand L is 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L1) substituted at the 4 position of the pyrazole ring by Cl (L2), Br (L3), or I (L4) and X = Cl- and I-] were synthesized and characterized using various analytical techniques. Complexes 1 and 3 were also characterized by single-crystal X-ray crystallography, and they crystallized as a monoclinic crystal system in space groups P21/n and P21/c, respectively. The complexes display good solution stability at physiological pH 7.4. The iodido-coordinated pyrazolylbenzimidazole ruthenium(II) p-cymene complexes (2, 4, 6, and 8) are more resistant toward hydrolysis and have less tendency to form monoaquated complexes in comparison to their chlorido analogues (1, 3, 5, and 7). The halido-substituted 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole ligands, designed as organic-directing molecules, inhibit vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation. In addition, the ruthenium(II) complexes display a potential to bind to DNA bases. The cytotoxicity profile of the complexes (IC50 ca. 9-12 µM for 4-8) against the triple-negative breast cancer cells (MDA-MB-231) show that most of the complexes are efficient. The lipophilicity and cellular accumulation data of the complexes show a good correlation with the cytotoxicity profile of 1-8. The representative complexes 3 and 7 demonstrate the capability of arresting the cell cycle in the G2/M phase and induce apoptosis. The inhibition of VEGFR2 phosphorylation with the representative ligands L2 and L4 and the corresponding metal complexes 3 and 7 in vitro shows that the organic-directing ligands and their complexes inhibit VEGFR2 phosphorylation. Besides, L2, L4, 3, and 7 inhibit the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and proto-oncogene tyrosine-protein kinase (Src), capable of acting downstream of VEGFR2 as well as independently. Compounds L2, L4, 3, and 7 have a lesser effect on ERK1/2 and more prominently affect Src phosphorylation. We extended the study for L2 and 3 in the Tg(fli1:gfp) zebrafish model and found that L2 is more effective in vivo compared to 3 in inhibiting angiogenesis.

Death From Primary Amebic Meningoencephalitis After Recreational Water Exposure During Recent Travel to India-Santa Clara County, California, 2020.

BACKGROUND: In February 2020, a man returned to the United States after an 11-day trip to India and died of primary amebic meningoencephalitis (PAM), caused by nasal exposure to the free-living ameba Naegleria fowleri found in warm water. We identified potential exposures, confirmed etiology, and described the molecular epidemiology of the infection. METHODS: We reviewed medical records to describe his clinical course and interviewed his family to determine water exposures. Genotyping was performed on the N. fowleri strain and compared with North American strains through repetitive nonpolymorphic nuclear loci analysis to identify differences. We reviewed N. fowleri strains in the National Center for Biotechnology Information database (GenBank) to determine genotypes present in India. RESULTS: The patient became acutely encephalopathic 3 days after returning; the only known nasal water exposure was at an indoor swimming pool in India 5 days earlier. Cerebrospinal fluid (CSF) testing demonstrated neutrophil-predominant pleocytosis and low glucose, but negative gram stain and culture. CSF microscopy revealed trophozoites; N. fowleri was detected by real-time polymerase chain reaction. Classical genotyping confirmed genotype I, common in the United States and among Indian strains in GenBank. The North American N. fowleri strains and the patient's strain varied at 5 nonpolymorphic loci. CONCLUSIONS: A man died from PAM after likely exposure at a vacation rental pool in India. We recommend including PAM in the differential diagnosis when CSF studies suggest bacterial meningitis but gram stain is negative. Genotyping can advance our understanding of N. fowleri molecular epidemiology and support future investigations.

Comparative Genomic and Transcriptomic Analysis of Naegleria fowleri Clinical and Environmental Isolates.

Out of over 40 species of Naegleria, which are free-living thermophilic amebae found in freshwater and soil worldwide, only Naegleria fowleri infects humans, causing primary amebic meningoencephalitis (PAM), a typically fatal brain disease. To understand the population structure of Naegleria species and the genetic relationships between N. fowleri isolates and to detect pathogenic factors, we characterized 52 novel clinical and environmental N. fowleri genomes and a single Naegleria lovaniensis strain, along with transcriptomic data for a subset of 37 N. fowleri isolates. Whole-genome analysis of 56 isolates from three Naegleria species (N. fowleri, N. lovaniensis, and Naegleria gruberi) identified several genes unique to N. fowleri that have previously been linked to the pathogenicity of N. fowleri, while other unique genes could be associated with novel pathogenicity factors in this highly fatal pathogen. Population structure analysis estimated the presence of 10 populations within the three Naegleria species, of which 7 populations were within N. fowleri. The whole-nuclear-genome (WNG) phylogenetic analysis showed an overall geographical clustering of N. fowleri isolates, with few exceptions, and provided higher resolution in identifying potential clusters of isolates beyond that of the traditional locus typing. There were only 34 genes that showed significant differences in gene expression between the clinical and environmental isolates. Genomic data generated in this study can be used for developing rapid molecular assays and to conduct future population-based global genomic analysis and will also be a valuable addition to genomic reference databases, where shotgun metagenomics data from routine water samples could be searched for the presence of N. fowleri strains. IMPORTANCE N. fowleri, the only known Naegleria species to infect humans, causes fatal brain disease. PAM cases from 1965 to 2016 showed <20 cases per year globally. Out of approximately 150 cases in North America since 1962, only four PAM survivors are known, yielding a >97% case fatality rate, which is critically high. Although the pathogenesis of N. fowleri has been studied for the last 50 years, pathogenetic factors that lead to human infection and breaching the blood-brain barrier remain unknown. In addition, little is known regarding the genomic diversity both within N. fowleri isolates and among Naegleria species. In this study, we generated novel genome sequences and performed comparative genomic and transcriptomic analysis of a set of 52 N. fowleri draft genome sequences from clinical and environmental isolates derived from all over the world in the last 53 years, which will help shape future genome-wide studies and develop sensitive assays for routine surveillance.

Draft Chromosome Sequences of a Clinical Isolate of the Free-Living Ameba Naegleria fowleri.

We present the chromosome sequences of a Naegleria fowleri isolate from a human primary amebic meningoencephalitis (PAM) case. The genome sequences were assembled from Illumina HiSeq and PacBio sequencing data and verified with the optical mapping data. This led to the identification of 37 contigs representing 37 chromosomes in N. fowleri.

Granulomatous amoebic encephalitis caused by Acanthamoeba in a patient with AIDS: a challenging diagnosis.

INTRODUCTION: Acanthamoeba spp. is a ubiquitous free-living amoeba that causes human infections affecting predominantly the cornea and central nervous system. The diagnosis and treatment of Acanthamoeba encephalitis is very challenging. CASE SUMMARY: A 53-year-old male with HIV/AIDS was admitted for altered mental status and fever. On initial examination, he had left hemianopia with left-sided weakness and numbness. MRI revealed an inflammatory and enhancing parenchymal mass associated with leptomeningeal enhancement in the occipitoparietal lobe containing multiple punctate hemorrhages. He was treated with empiric antibiotics for presumptive toxoplasmosis, brain abscess, fungal infection and tuberculosis with an unremarkable lymphoma work up. Initial brain biopsy studies were unremarkable except for non-specific granulomas and adjacent necrotic tissue. The patient passed away 2.5 months after initial presentation with no diagnosis. Post-mortem testing by the Centers for Disease Control and Prevention (CDC) confirmed the diagnosis of granulomatous amoebic encephalitis (GAE) by visualization with immunohistochemistry staining and PCR. Recovery is rare from GAE likely due to delay in diagnosis. CONCLUSIONS: This case illustrates the importance of including GAE into the differential diagnosis of brain mass. We advocate early molecular testing of tissue specimen by the CDC to achieve an appropriate diagnosis, and a multidisciplinary approach for the management of this condition.

Diagnostic evaluation of fatal Balamuthia mandrillaris meningoencephalitis in a captive Bornean orangutan (Pongo pygmaeus) with identification of potential environmental source and evidence of chronic exposure.

A female Bornean orangutan (Pongo pygmaeus) aged 11 years and 6 months was examined by veterinarians after caretakers observed lethargy and facial grimacing. Within 72 h the primate had left-sided hemiparesis that worsened over the next week. An MRI revealed a focal right-sided cerebral mass suspected to be a neoplasm. Ten days after onset of clinical signs, the orangutan died. On postmortem exam, the medial right parietal lobe was replaced by a 7 × 4 × 3.5 cm focus of neuromalacia and hemorrhage that displaced the lateral ventricle and abutted the corpus callosum. Histopathology of the cerebral lesion revealed pyogranulomatous meningoencephalitis with intralesional amoeba trophozoites and rare cysts. Fresh parietal lobe was submitted to the Centers for Disease Control and Prevention lab for multiplex free-living amoebae real-time PCR and detected Balamuthia mandrillaris DNA at a high burden. Mitochondrial DNA was sequenced, and a 760-bp locus 19443F/20251R was compared to several human infections of B. mandrillaris and shown to be identical to the isolates from four human cases of encephalitis: 1998 in Australia, 1999 in California, 2000 in New York, and 2010 in Arizona. Indirect immunofluorescent antibody testing of stored serum samples indicated exposure to B. mandrillaris for at least 2 years prior to death. Within 1 week of the orangutan's death, water from the exhibit was analyzed and identified the presence of B. mandrillaris DNA, elucidating a possible source of exposure. B. mandrillaris, first reported in a mandrill in 1986, has since occurred in humans and animals and is now considered an important emerging pathogen.

A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans.

There are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

HMG-CoA Reductase Inhibitors as Drug Leads against Naegleria fowleri.

Primary amebic meningoencephalitis (PAM), caused by the free-living ameba Naegleria fowleri, has a fatality rate of over 97%. Treatment of PAM relies on amphotericin B in combination with other drugs, but few patients have survived with the existing drug treatment regimens. Therefore, development of effective drugs is a critical unmet need to avert deaths from PAM. Since ergosterol is one of the major sterols in the membrane of N. fowleri, disruption of isoprenoid and sterol biosynthesis by small-molecule inhibitors may be an effective intervention strategy against N. fowleri. The genome of N. fowleri contains a gene encoding HMG-CoA reductase (HMGR); the catalytic domains of human and N. fowleri HMGR share <60% sequence identity with only two amino acid substitutions in the active site of the enzyme. Considering the similarity of human and N. fowleri HMGR, we tested well-tolerated and widely used HMGR inhibitors, known as cholesterol-lowering statins, against N. fowleri. We identified blood-brain-barrier-permeable pitavastatin as a potent amebicidal agent against the U.S., Australian, and European strains of N. fowleri. Pitavastatin was equipotent to amphotericin B against the European strain of N. fowleri; it killed about 80% of trophozoites within 16 h of drug exposure. Pretreatment of trophozoites with mevalonate, the product of HMGR, rescued N. fowleri from inhibitory effects of statins, demonstrating that HMGR of N. fowleri is the target of statins. Because of the good safety profile and availability for both adult and pediatric uses, consideration should be given to repurposing the fast-acting pitavastatin for the treatment of PAM.

Rapid, Noninvasive Diagnosis of Balamuthia mandrillaris Encephalitis by a Plasma-Based Next-Generation Sequencing Test.

Granulomatous amoebic encephalitis (GAE) caused by Balamuthia mandrillaris is a rare subacute infection with exceptionally high mortality. Diagnosis is typically made by brain biopsy or at autopsy. Detection of Balamuthia mandrillaris cell-free DNA by next-generation sequencing of plasma enabled rapid, noninvasive diagnosis in a case of amoebic encephalitis.

Identification of Entamoeba histolytica in Patients with Suspected Amebiasis in Jordan Using PCR-based Assays.

BACKGROUND AND OBJECTIVE: Identification of Entamoeba histolytica (E. histolytica) by microscopy alone can be problematic because E. dispar and E. moshkovskii are morphologically similar to E. histolytica. Therefore, this study aimed to assess the performance of microscopy in the detection of E. histolytica in stool specimens with the help of PCR-based assays and enzyme-linked immunosorbent assay (ELISA). MATERIALS AND METHODS: Between September, 2017 and September, 2018, 200 stool specimens were obtained from Jordanian patients with suspected amebiasis. All specimens were subjected to microscopic analysis. DNA was extracted from the microscopy-positive stool samples. A conventional PCR and a duplex real-time PCR were performed to detect E. histolytica and E. dispar. RESULTS: By microscopy, 35% (70/200) of specimens were tested positive for Entamoeba complex. All 70 microscopic-positive Entamoeba complex samples were negative for the presence of E. histolytica by the NOVITEC® E. histolytica ELISA assay. All 70 samples positive by microscopy were negative for the presence of E. histolytica and E. dispar by PCR-based assays. CONCLUSION: We suspect some of these microscopy-positive stool specimens might contain a potentially novel species of Entamoeba that could not be detected by ELISA or PCR-based assays specific for E. histolytica and E. dispar. Diagnosis of amebiasis remains challenging here in Jordan and hence highlighting the need for improved diagnostic method.

A case of primary amebic meningoencephalitis caused by Naegleria fowleri in Bangladesh.

We present the first recognized case of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri in a 15-year-old male from Bangladesh. He performed daily nasal rinsing with untreated ground water and bathed in untreated ground water or river water, which likely exposed him to N. fowleri.

Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol.

Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2-1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.