Ultrasensitive malaria detection system for Anopheles mosquito field surveillance using droplet digital PCR.

Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques. A total of 152 mosquitoes from six anopheline species were collected and first analyzed by a standard 18S rRNA nested-PCR analysis for malaria parasite which yielded negative results in all collected mosquitoes. Later, ddPCR was used and could detect simian malaria parasite DNA, i.e. Plasmodium cynomolgi, in 25 collected mosquitoes. And this is the first report of simian malaria parasite DNA detection in Anopheles sawadwongporni. This finding proves that ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles mosquitoes and can expand our understanding of the zoonotic potential of malaria transmission between monkeys and humans.

Corrigendum: Regeneration of the cerebral cortex by direct chemical reprogramming of macrophages into neuronal cells in acute ischemic stroke.

[This corrects the article DOI: 10.3389/fncel.2023.1225504.].

Postmortem Identification of Genetic Variations Associated with Sudden Unexpected Death in Young People.

Sudden unexpected death in the young (SUDY) is a traumatic occurrence for their family; however, information on the genetic variations associated with the condition is currently lacking. It is important to carry out postmortem genetic analyses in cases of sudden death to provide information for relatives and to allow appropriate genetic counselling and clinical follow-up. This study aimed to investigate the genetic variations associated with the occurrence of SUDY in Japan, using next-generation sequencing (NGS). The study included 18 cases of SUDY (16 males, 2 females; age 15-47 years) who underwent autopsy, including NGS DNA sequencing for molecular analysis. A total of 168 genes were selected from the sequencing panel and filtered, resulting in the identification of 60 variants in cardiac disease-related genes. Many of the cases had several of these genetic variants and some cases had a cardiac phenotype. The identification of genetic variants using NGS provides important information regarding the pathogenicity of sudden death.

Missense mutation of NRAS is associated with malignant progression in neurocutaneous melanosis.

Neurocutaneous melanosis (NCM) is a rare congenital neurocutaneous syndrome characterized by congenital melanocytic nevus of skin and abnormal proliferation of leptomeningeal melanocytes. Early acquisition of post-zygotic somatic mutations has been postulated to underlie the pathogenesis of NCM. The pathogenesis of NCM remains to be fully elucidated, and treatment options have not been established. Here, we report for the first time, multiregional genomic analyses in a 3-year-old autopsied girl with leptomeningeal melanomatosis associated with NCM, in which a ventriculo-peritoneal (VP) shunt was inserted for the treatment of hydrocephalus. The patient expired six months after the onset due to respiratory failure caused by abdominal dissemination via VP shunt. We performed multiregional exome sequencing to identify genomic differences among brain and abdominal tumors, nevus, and normal tissues. A total of 87 somatic mutations were found in 71 genes, with a significantly large number of gene mutations found in the tumor site. The genetic alterations detected in the nevus were only few and not shared with other sites. Three mutations, namely GNAQ R183Q, S1PR3 G89S and NRAS G12V, considered pathogenic, were found, although S1PR3 mutations have not been previously reported in melanocytic tumors. GNAQ and S1PR3 mutations were shared in both tumor and normal sites. Moreover, the mutant allele frequencies of the two mutations were markedly higher in tumor sites than in normal sites, with copy-neutral loss-of-heterozygosity (CN-LOH) occurring in tumor. NRAS mutation was found only in the abdominal tumor and was thought to be responsible for malignant progression in the present case. Multiregional comprehensive genetic analysis may lead to discovering novel driver mutations associated with tumorigenesis and targeted therapy.

Reliable detection of genetic alterations in cyst fluid DNA for the diagnosis of brain tumors.

PURPOSE: Liquid biopsy of cyst fluid in brain tumors has not been extensively studied to date. The present study was performed to see whether diagnostic genetic alterations found in brain tumor tissue DNA could also be detected in cell-free DNA (cfDNA) of cyst fluid in cystic brain tumors. METHODS: Cyst fluid was obtained from 22 patients undergoing surgery for a cystic brain tumor with confirmed genetic alterations in tumor DNA. Pathological diagnoses based on WHO 2021 classification and diagnostic alterations in the tumor DNA, such as IDH1 R132H and TERT promoter mutation for oligodendrogliomas, were detected by Sanger sequencing. The same alterations were analyzed by both droplet digital PCR (ddPCR) and Sanger sequencing in cyst fluid cfDNA. Additionally, multiplex ligation-dependent probe amplification (MLPA) assays were performed to assess 1p/19q status, presence of CDKN2A loss, PTEN loss and EGFR amplification, to assess whether differentiating between astrocytomas and oligodendrogliomas and grading is possible from cyst fluid cfDNA. RESULTS: Twenty-five genetic alterations were found in 22 tumor samples. All (100%) alterations were detected in cyst fluid cfDNA by ddPCR. Twenty of the 25 (80%) alterations were also detected by Sanger sequencing of cyst fluid cfDNA. Variant allele frequency (VAF) in cyst fluid cfDNA was comparable to that of tumor DNA (R = 0.62, Pearson's correlation). MLPA was feasible in 11 out of 17 (65%) diffuse gliomas, with close correlation of results between tumor DNA and cyst fluid cfDNA. CONCLUSION: Cell-free DNA obtained from cyst fluid in cystic brain tumors is a reliable alternative to tumor DNA when diagnosing brain tumors.

Regeneration of the cerebral cortex by direct chemical reprogramming of macrophages into neuronal cells in acute ischemic stroke.

Theoretically, direct chemical reprogramming of somatic cells into neurons in the infarct area represents a promising regenerative therapy for ischemic stroke. Previous studies have reported that human fibroblasts and astrocytes transdifferentiate into neuronal cells in the presence of small molecules without introducing ectopic transgenes. However, the optimal combination of small molecules for the transdifferentiation of macrophages into neurons has not yet been determined. The authors hypothesized that a combination of small molecules could induce the transdifferentiation of monocyte-derived macrophages into neurons and that the administration of this combination may be a regenerative therapy for ischemic stroke because monocytes and macrophages are directly involved in the ischemic area. Transcriptomes and morphologies of the cells were compared before and after stimulation using RNA sequencing and immunofluorescence staining. Microscopic analyses were also performed to identify cell markers and evaluate functional recovery by blinded examination following the administration of small molecules after ischemic stroke in CB-17 mice. In this study, an essential combination of six small molecules [CHIR99021, Dorsomorphin, Forskolin, isoxazole-9 (ISX-9), Y27632, and DB2313] that transdifferentiated monocyte-derived macrophages into neurons in vitro was identified. Moreover, administration of six small molecules after cerebral ischemia in model animals generated a new neuronal layer in the infarct cortex by converting macrophages into neuronal cells, ultimately improving neurological function. These results suggest that altering the transdifferentiation of monocyte-derived macrophages by the small molecules to adjust their adaptive response will facilitate the development of regenerative therapies for ischemic stroke.

TDP-43 differentially propagates to induce antero- and retrograde degeneration in the corticospinal circuits in mouse focal ALS models.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by TDP-43 inclusions in the cortical and spinal motor neurons. It remains unknown whether and how pathogenic TDP-43 spreads across neural connections to progress degenerative processes in the cortico-spinal motor circuitry. Here we established novel mouse ALS models that initially induced mutant TDP-43 inclusions in specific neuronal or cell types in the motor circuits, and investigated whether TDP-43 and relevant pathological processes spread across neuronal or cellular connections. We first developed ALS models that primarily induced TDP-43 inclusions in the corticospinal neurons, spinal motor neurons, or forelimb skeletal muscle, by using adeno-associated virus (AAV) expressing mutant TDP-43. We found that TDP-43 induced in the corticospinal neurons was transported along the axons anterogradely and transferred to the oligodendrocytes along the corticospinal tract (CST), coinciding with mild axon degeneration. In contrast, TDP-43 introduced in the spinal motor neurons did not spread retrogradely to the cortical or spinal neurons; however, it induced an extreme loss of spinal motor neurons and subsequent degeneration of neighboring spinal neurons, suggesting a degenerative propagation in a retrograde manner in the spinal cord. The intraspinal degeneration further led to severe muscle atrophy. Finally, TDP-43 induced in the skeletal muscle did not propagate pathological events to spinal neurons retrogradely. Our data revealed that mutant TDP-43 spread across neuro-glial connections anterogradely in the corticospinal pathway, whereas it exhibited different retrograde degenerative properties in the spinal circuits. This suggests that pathogenic TDP-43 may induce distinct antero- and retrograde mechanisms of degeneration in the motor system in ALS.

Bleeding-Source Exploration in Subdural Hematoma: Observational Study on the Usefulness of Postmortem Computed Tomography Angiography.

In a few cases, postmortem computed tomography angiography (PMCTA) is effective in postmortem detection of cortical artery rupture causing subdural hematoma (SDH), which is difficult to detect at autopsy. Here, we explore the usefulness and limitations of PMCTA in detecting the sites of cortical arterial rupture for SDH. In 6 of 10 cases, extravascular leakage of contrast material at nine different places enabled PMCTA to identify cortical arterial rupture. PMCTA did not induce destructive arterial artifacts, which often occur during autopsy. We found that, although not in all cases, PMCTA could show the site of cortical arterial rupture causing subdural hematoma in some cases. This technique is beneficial for cases of SDH autopsy, as it can be performed nondestructively and before destructive artifacts from the autopsy occur.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD.

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Phosphorylation of α-synuclein at T64 results in distinct oligomers and exerts toxicity in models of Parkinson's disease.

α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.

Detection and Morphological Analysis of Micro-Ruptured Cortical Arteries in Subdural Hematoma: Three-Dimensional Visualization Using the Tissue-Clearing Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational Analysis Method.

One of the causes of bleeding in subdural hematoma is cortical artery rupture, which is difficult to detect at autopsy. Therefore, reports of autopsy cases with this condition are limited and hence, the pathogenesis of subdural hematoma remains unclear. Herein, for the detection and morphological analysis of cortical artery ruptures as the bleeding sources of subdural hematoma, we used the tissue-clearing CUBIC (clear, unobstructed, brain/body imaging cocktails and computational analysis) method with light-sheet fluorescence microscopy and reconstructed the two-dimensional and three-dimensional images. Using the CUBIC method, we could clearly visualize and detect cortical artery ruptures that were missed by conventional methods. Indeed, the CUBIC method enables three-dimensional morphological analysis of cortical arteries including the ruptured area, and the creation of cross-sectional two-dimensional images in any direction, which are similar to histopathological images. This highlights the effectiveness of the CUBIC method for subdural hematoma analysis.

Endogenous human retrovirus-K is not increased in the affected tissues of Japanese ALS patients.

Activation of human endogenous retrovirus-K (HERV-K) is one of the proposed risk factors for amyotrophic lateral sclerosis (ALS). The HERV-K envelope protein has been reported to show neurotoxicity, and development of therapy with reverse transcriptase inhibitors is being investigated. On the other hand, some reports have failed to show HERV-K activation in ALS. In this study, we analyzed the expression of HERV-K mRNA in the motor cortex and spinal cord of 15 Japanese patients with sporadic ALS and 19 controls using reverse transcriptase droplet digital PCR. This revealed no significant increase of HERV-K expression in ALS-affected tissues, suggesting that the association between ALS and HERV-K remains questionable.

PROS1 variant in sudden death case of pulmonary embolism caused by calcification in the inferior vena cava: The importance of postmortem genetic analysis.

A Japanese man in his 30s died suddenly. Postmortem computed tomography and autopsy revealed a pulmonary embolism from an organizing thrombus in the inferior vena cava as the cause of death. Genomic analysis of congenital thrombophilia-related genes (i.e., SERPINC1, PROC, PROS1, F2, F5, PLG, and MTHFR) revealed a heterozygous variant of PROS1 (p.A139V), which has been reported in patients with congenital protein S deficiency. After a genetic conference that included forensic pathologists, molecular scientists, genetic researchers, genetic clinicians, and clinical physicians, the results of the genetic analysis were explained to the family. Biochemical analyses of protein S (PS) activity and total PS antigen levels were performed with samples from the deceased's family and genetic analysis was not performed until clinical symptoms appear. Herein we demonstrate the importance of genetic background in cases of a sudden death due to pulmonary embolism.

Candesartan prevents arteriopathy progression in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy model.

Cerebral small vessel disease (CSVD) causes dementia and gait disturbance due to arteriopathy. Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary form of CSVD caused by loss of high-temperature requirement A1 (HTRA1) serine protease activity. In CARASIL, arteriopathy causes intimal thickening, smooth muscle cell (SMC) degeneration, elastic lamina splitting, and vasodilation. The molecular mechanisms were proposed to involve the accumulation of matrisome proteins as substrates or abnormalities in transforming growth factor ß (TGF-ß) signaling. Here, we show that HTRA1-/- mice exhibited features of CARASIL-associated arteriopathy: intimal thickening, abnormal elastic lamina, and vasodilation. In addition, the mice exhibited reduced distensibility of the cerebral arteries and blood flow in the cerebral cortex. In the thickened intima, matrisome proteins, including the hub protein fibronectin (FN) and latent TGF-ß binding protein 4 (LTBP-4), which are substrates of HTRA1, accumulated. Candesartan treatment alleviated matrisome protein accumulation and normalized the vascular distensibility and cerebral blood flow. Furthermore, candesartan reduced the mRNA expression of Fn1, Ltbp-4, and Adamtsl2, which are involved in forming the extracellular matrix network. Our results indicate that these accumulated matrisome proteins may be potential therapeutic targets for arteriopathy in CARASIL.

Corrigendum: HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade.

[This corrects the article DOI: 10.3389/fneur.2019.00693.].

The usefulness of postmortem computed tomography angiography for subdural hematoma caused by rupture of the cortical artery: A report of two autopsy cases and a literature review.

Acute subdural hematoma (SDH) occurs following severe head trauma with brain contusion or rupture of bridging veins. Conversely, SDH caused by rupture of a cortical artery without trauma or with minor trauma is also possible. Although over 180 cases of the latter SDH have been reported, they were predominantly diagnosed only during surgery, and therefore, no adequate histological evaluation has been performed. Therefore, essential etiology of this SDH type has remained unclear. In addition, the scarcity of autopsy cases may be attributed to arterial rupture being missed if the microscopic findings are too minimal to detect during autopsy. Here, we describe two autopsy cases of SDH of cortical artery origin. Extravasation on postmortem computed tomography angiography and arterial leakage on macroscopic observation during autopsy facilitated detection of the ruptured artery and allowed detailed histological evaluation of the ruptured artery and adjacent dura mater. The etiology of arterial rupture is briefly described on the basis of histopathological findings in this study and the available literature.

HTRA1-Related Cerebral Small Vessel Disease: A Review of the Literature.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is clinically characterized by early-onset dementia, stroke, spondylosis deformans, and alopecia. In CARASIL cases, brain magnetic resonance imaging reveals severe white matter hyperintensities (WMHs), lacunar infarctions, and microbleeds. CARASIL is caused by a homozygous mutation in high-temperature requirement A serine peptidase 1 (HTRA1). Recently, it was reported that several heterozygous mutations in HTRA1 also cause cerebral small vessel disease (CSVD). Although patients with heterozygous HTRA1-related CSVD (symptomatic carriers) are reported to have a milder form of CARASIL, little is known about the clinical and genetic differences between the two diseases. Given this gap in the literature, we collected clinical information on HTRA1-related CSVD from a review of the literature to help clarify the differences between symptomatic carriers and CARASIL and the features of both diseases. Forty-six symptomatic carriers and 28 patients with CARASIL were investigated. Twenty-eight mutations in symptomatic carriers and 22 mutations in CARASIL were identified. Missense mutations in symptomatic carriers are more frequently identified in the linker or loop 3 (L3)/loop D (LD) domains, which are critical sites in activating protease activity. The ages at onset of neurological symptoms/signs were significantly higher in symptomatic carriers than in CARASIL, and the frequency of characteristic extraneurological findings and confluent WMHs were significantly higher in CARASIL than in symptomatic carriers. As previously reported, heterozygous HTRA1-related CSVD has a milder clinical presentation of CARASIL. It seems that haploinsufficiency can cause CSVD among symptomatic carriers according to the several patients with heterozygous nonsense/frameshift mutations. However, the differing locations of mutations found in the two diseases indicate that distinct molecular mechanisms influence the development of CSVD in patients with HTRA1-related CSVD. These findings further support continued careful examination of the pathogenicity of mutations located outside the linker or LD/L3 domain in symptomatic carriers.

HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade.

Background: Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Most carriers for HTRA1 mutations are asymptomatic, but more than 10 mutations have been reported in symptomatic carriers. The molecular differences between the mutations identified in symptomatic carriers and mutations identified only in CARASIL patients are unclear. HTRA1 is a serine protease that forms homotrimers, with each HTRA1 subunit activating the adjacent HTRA1 via the sensor domain of loop 3 (L3) and the activation domain of loop D (LD). Previously, we analyzed four HTRA1 mutant proteins identified in symptomatic carriers and found that they were unable to form trimers or had mutations in the LD or L3 domain. The mutant HTRA1s with these properties are presumed to inhibit trimer-dependent activation cascade. Indeed, these mutant HTRA1s inhibited wild-type (WT) protease activity. In this study, we further analyzed 15 missense HTRA1s to clarify the molecular character of mutant HTRA1s identified in symptomatic carriers. Methods: We analyzed 12 missense HTRA1s identified in symptomatic carriers (hetero-HTRA1) and three missense HTRA1s found only in CARASIL (CARASIL-HTRA1). The protease activity of the purified recombinant mutant HTRA1s was measured using fluorescein isothiocyanate-labeled casein as substrate. Oligomeric structure was evaluated by size-exclusion chromatography. The protease activities of mixtures of WT with each mutant HTRA1 were also measured. Results: Five hetero-HTRA1s had normal protease activity and were excluded from further analysis. Four of the seven hetero-HTRA1s and one of the three CARASIL-HTRA1s were unable to form trimers. The other three hetero-HTRA1s had mutations in the LD domain. Together with our previous work, 10 of 11 hetero-HTRA1s and two of six CARASIL-HTRA1s were either defective in trimerization or had mutations in the LD or L3 domain (P = 0.006). By contrast, eight of 11 hetero-HTRA1s and two of six CARASIL-HTRA1 inhibited WT protease activity (P = 0.162). Conclusions: HTRA1 mutations identified in symptomatic carriers have the property of interfering the trimer-dependent activation cascade of HTRA1.

Non-genetically modified models exhibit TARDBP mRNA increase due to perturbed TDP-43 autoregulation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by accumulation of fragmented insoluble TDP-43 and loss of TDP-43 from the nucleus. Increased expression of exogenous TARDBP (encoding TDP-43) induces TDP-43 pathology and cytotoxicity, suggesting the involvement of aberrant expression of TDP-43 in the pathogenesis of ALS. In normal conditions, however, the amount of TDP-43 is tightly regulated by the autoregulatory mechanism involving alternative splicing of TARDBP mRNA. To investigate the influence of autoregulation dysfunction, we inhibited the splicing of cryptic intron 6 using antisense oligonucleotides in vivo. This inhibition doubled the Tardbp mRNA expression, increased the fragmented insoluble TDP-43, and reduced the number of motor neurons in the mouse spinal cord. In human induced pluripotent stem cell-derived neurons, the splicing inhibition of intron 6 increased TARDBP mRNA and decreased nuclear TDP-43. These non-genetically modified models exhibiting rise in the TARDBP mRNA levels suggest that TDP-43 autoregulation turbulence might be linked to the pathogenesis of ALS.