Lotusine ameliorates propionic acid-induced autism spectrum disorder-like behavior in mice by activating D1 dopamine receptor in medial prefrontal cortex.

Autism spectrum disorder (ASD) is a multifaceted neuropsychiatric condition for which effective drug therapy for core clinical symptoms remains elusive. Lotusine, known for its neuroprotective properties in the treatment of neurological disorders, holds potential in addressing ASD. Nevertheless, its specific efficacy in ASD remains uncertain. This study aims to investigate the therapeutic potential of lotusine in ASD and elucidate the underlying molecular mechanisms. We induced an ASD mouse model through intracerebroventricular-propionic acid (ICV-PPA) injection for 7 days, followed by lotusine administration for 5 days. The efficacy of lotusine was evaluated through a battery of behavioral tests, including the three-chamber social test. The underlying mechanisms of lotusine action in ameliorating ASD-like behavior were investigated in the medial prefrontal cortex (mPFC) using whole-cell patch-clamp recordings, western blotting, immunofluorescence staining, molecular docking, and cellular thermal shift assay. The efficacy and mechanisms of lotusine were further validated in vitro. Lotusine effectively alleviated social deficits induced by ICV-PPA injection in mice by counteracting the reduction in miniature excitatory postsynaptic current frequency within the mPFC. Moreover, lotusine enhanced neuronal activity and ameliorated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor dysfunction in ICV-PPA infusion mice by upregulating c-fos, p-GluA1 Ser 845, and p-GluA1 Ser 831 protein levels within the mPFC. Our findings also suggest that lotusine may exert its effects through modulation of the D1 dopamine receptor (DRD1). Furthermore, the rescuing effects of lotusine were nullified by a DRD1 antagonist in PC12 cells. In summary, our results revealed that lotusine ameliorates ASD-like behavior through targeted modulation of DRD1, ultimately enhancing excitatory synaptic transmission. These findings highlight the potential of lotusine as a nutritional supplement in the treatment of ASD.

Evolutionary History and Genetic Variation of Zika Virus: Connection Between Thailand Zika Viruses and Global Outbreaks Strains.

Background: Zika virus (ZIKV) has significant potential to cause future outbreaks due to insufficient countermeasures. The evolution of ZIKV in Southeast Asian countries remains poorly understood. Materials and Methods: The phylogenetic, phylogeographic network, and recombination analyses of 366 ZIKV complete genome sequences identified between 1947 and 2021 were performed and the amino acid variation landscape was determined to reveal the evolutionary characteristics. Results: ZIKV falls into two major genogroups: GI and GII, segregated into further subgenogroups (GI-1 to GI-3) and (GII-1 to GII-3), respectively. Importantly, Thailand strains cluster with Southeast Asian outbreak strains (Singapore 2016, the Philippines 2012, Cambodia 2010) into GII-2 and form a lineage independent of French Polynesia and the Americas large outbreak strains. Thailand ZIKV strains shared their ancestral route to the strains from French Polynesia, which further connects to Brazil ZIKV through a short mutational branch. Both recombination and specific mutations may contribute to the emergence of new virus lineage in Thailand. Conclusion: This report provides insights into the evolutionary characteristics of ZIKV in Southeast Asia, which may be helpful for epidemiological investigation, vaccine development, and surveillance of the virus.

Bioinformatics Analysis of MSH1 Genes of Green Plants: Multiple Parallel Length Expansions, Intron Gains and Losses, Partial Gene Duplications, and Alternative Splicing.

MutS homolog 1 (MSH1) is involved in the recombining and repairing of organelle genomes and is essential for maintaining their stability. Previous studies indicated that the length of the gene varied greatly among species and detected species-specific partial gene duplications in Physcomitrella patens. However, there are critical gaps in the understanding of the gene size expansion, and the extent of the partial gene duplication of MSH1 remains unclear. Here, we screened MSH1 genes in 85 selected species with genome sequences representing the main clades of green plants (Viridiplantae). We identified the MSH1 gene in all lineages of green plants, except for nine incomplete species, for bioinformatics analysis. The gene is a singleton gene in most of the selected species with conserved amino acids and protein domains. Gene length varies greatly among the species, ranging from 3234 bp in Ostreococcus tauri to 805,861 bp in Cycas panzhihuaensis. The expansion of MSH1 repeatedly occurred in multiple clades, especially in Gymnosperms, Orchidaceae, and Chloranthus spicatus. MSH1 has exceptionally long introns in certain species due to the gene length expansion, and the longest intron even reaches 101,025 bp. And the gene length is positively correlated with the proportion of the transposable elements (TEs) in the introns. In addition, gene structure analysis indicated that the MSH1 of green plants had undergone parallel intron gains and losses in all major lineages. However, the intron number of seed plants (gymnosperm and angiosperm) is relatively stable. All the selected gymnosperms contain 22 introns except for Gnetum montanum and Welwitschia mirabilis, while all the selected angiosperm species preserve 21 introns except for the ANA grade. Notably, the coding region of MSH1 in algae presents an exceptionally high GC content (47.7% to 75.5%). Moreover, over one-third of the selected species contain species-specific partial gene duplications of MSH1, except for the conserved mosses-specific partial gene duplication. Additionally, we found conserved alternatively spliced MSH1 transcripts in five species. The study of MSH1 sheds light on the evolution of the long genes of green plants.

A pathogenesis-related protein 1 of Cucurbita moschata responds to powdery mildew infection.

Pumpkin (Cucurbita moschata Duch.) productivity is severely hindered by powdery mildew (PM) worldwide. The causative agent of pumpkin PM is Podosphaera xanthii, a biotrophic fungus. Pathogenesis-related protein 1 (PR1) homolog was previously identified from transcriptomic analysis of a PM-resistant pumpkin. Here, we investigated the effects of CmPR1 gene from pumpkin for resistance to PM. Subcellular localization assay revealed that CmPR1 is a cytoplasmic protein in plants. The expression of CmPR1 gene was strongly induced by P. xanthii inoculation at 48 h and exogenous ethylene (ET), jasmonic acid (JA) and NaCl treatments, but repressed by H2O2 and salicylic acid (SA) treatments. Visual disease symptoms, histological observations of fungal growth and host cell death, and accumulation of H2O2 in transgenic tobacco plants indicated that CmPR1 overexpression significantly enhanced the resistance to Golovinomyces cichoracearum compared to wild type plants during PM pathogens infection, possibly due to inducing cell death and H2O2 accumulation near infected sites. The expression of PR1a was significantly induced in transgenic tobacco plants in response to G. cichoracearum, suggesting that CmPR1 overexpression positively modulates the resistance to PM via the SA signaling pathway. These findings indicate that CmPR1 is a defense response gene in C. moschata and can be exploited to develop disease-resistant crop varieties.

Small Molecule Nitazoxanide Inhibits Osteogenic Differentiation and Promotes Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

OBJECTIVE: To investigate the potential effect of small molecule nitazoxanide (NTZ) on the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Cell counting Kit-8 assay was used to examine the effect of NTZ on proliferation of BMSCs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis were used to measure the expression of osteogenic and adipogenic marker gene. Alkaline phosphatase (ALP) staining and activity assay and Alizarin Red S (ARS) staining were used to investigate the effect of NTZ on osteogenesis. Oil red O (ORO) staining assay was used to assess the impact of NTZ on adipogenesis. RESULTS: NTZ significantly suppressed the osteogenic differentiation but promoted the adipogenic differentiation of BMSCs. Mechanistically, NTZ regulated osteogenic/adipogenic differentiation of BMSCs by inhibiting the Wnt/ß-catenin signalling pathway. The addition of Wnt/ß-catenin signalling pathway activator, lithium chloride, could reverse the effect of NTZ on BMSCs. CONCLUSION: NTZ affected osteogenic and adipogenic differentiation of BMSCs with the involvement of Wnt/ß-catenin signalling pathway. This finding expanded the understanding of NTZ pharmacology and indicated that NTZ might have an adverse effect on bone homeostasis.

Improvement of Learning and Memory by Elevating Brain D-Aspartate in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is an inherited human mental retardation that arises from expansion of a CGG repeat in the Fmr1 gene, causing loss of the fragile X mental retardation protein (FMRP). It is reported that N-methyl-D-aspartate receptor (NMDAR)-mediated facilitation of long-term potentiation (LTP) and fear memory are impaired in Fmr1 knockout (KO) mice. In this study, biological, pharmacological, and electrophysiological techniques were performed to determine the roles of D-aspartate (D-Asp), a modulator of NMDAR, and its metabolizing enzyme D-aspartate oxidase (DDO) in Fmr1 KO mice. Levels of D-Asp were decreased in the medial prefrontal cortex (mPFC ); however, the levels of its metabolizing enzyme DDO were increased. Electrophysiological recordings indicated that oral drinking of D-Asp recovered LTP induction in mPFC from Fmr1 KO mice. Moreover, chronic oral administration of D-Asp reversed behavioral deficits of cognition and locomotor coordination in Fmr1 KO mice. The therapeutic action of D-Asp was partially through regulating functions of NMDARs and mGluR5/mTOR/4E-BP signaling pathways. In conclusion, supplement of D-Asp may benefit for synaptic plasticity and behaviors in Fmr1 KO mice and offer a potential therapeutic strategy for FXS.

Phylogeographic dynamics and molecular characteristics of Enterovirus 71 in China.

Enterovirus 71 (EV71) and coxsackievirus (CV-A16) are the major etiological agents of hand, foot and mouth disease (HFMD). This report reviewed the full-length genomic sequences of EV71 identified in different provinces of China between 1998 and 2019 (a total of 312) in addition to eight worldwide reference genomes to address the genomic evolution and genetic events. The main prevalent EV71 strians in China are C4 genotypes, co-circulating with a few A, B5, C1, and C2 subgenotypes. A new emerging subgenotype in China was identified and classified as B6 genotype. Phylogeographic analysis revealed multiple branches, where a Jiangsu strain 2006-52-9 (GenBank ID: KP266579.1) was linked to different subgenotypes through multiple long mutant branches, including the CV-A16 viruses through the A genotype. Furthermore, identification of 28 natural recombination events suggests that the emergence of new genotypes are associated with intratypic recombination involving EV71 strains and intertypic recombination between EV71 and CV-A16 strains. Compared with the structural proteins, the non-structural proteins of EV71 seem to be highly variable with the highest variable regions of peptidase C3 (3C protein), P2A, and the N-terminus of RNA-dependent RNA polymerase. This study updates the phylogenetic and phylogeographic information of EV71 and provides clues to the emergence of new genotypes of EV71 based on genetics.

Genetic comparison of transmissible gastroenteritis coronaviruses.

Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that threatens animal health and remains elusive despite years of research efforts. The systematical analysis of all available full-length genomes of TGEVs (a total of 43) and porcine respiratory coronaviruses PRCVs (a total of 7) showed that TGEVs fell into two independent evolutionary phylogenetic clades, GI and GII. Viruses circulating in China (until 2021) clustered with the traditional or attenuated vaccine strains within the same evolutionary clades (GI). In contrast, viruses latterly isolated in the USA fell into GII clade. The viruses circulating in China have a lower similarity with that isolated latterly in the USA all through the viral genome. In addition, at least four potential genomic recombination events were identified, three of which occurred in GI clade and one in GII clade. TGEVs circulating in China are distinct from the viruses latterly isolated in the USA at either genomic nucleotide or antigenic levels. Genomic recombination serves as a factor driving the expansion of TGEV genomic diversity.

Multiple potential recombination events among Newcastle disease virus genomes in China between 1946 and 2020.

Introduction: Newcastle Disease Virus (NDV) is a highly adaptable virus with large genetic diversity that has been widely studied for its oncolytic activities and potential as a vector vaccine. This study investigated the molecular characteristics of 517 complete NDV strains collected from 26 provinces across China between 1946-2020. Methods: Herein, phylogenetic, phylogeographic network, recombination, and amino acid variability analyses were performed to reveal the evolutionary characteristics of NDV in China. Results and discussions: Phylogenetic analysis revealed the existence of two major groups: GI, which comprises a single genotype Ib, and GII group encompassing eight genotypes (I, II, III, VI. VII. VIII, IX and XII). The Ib genotype is found to dominate China (34%), particularly South and East China, followed by VII (24%) and VI (22%). NDV strains from the two identified groups exhibited great dissimilarities at the nucleotide level of phosphoprotein (P), matrix protein (M), fusion protein (F), and haemagglutinin-neuraminidase (HN) genes. Consistently, the phylogeographic network analysis revealed two main Network Clusters linked to a possible ancestral node from Hunan (strain MH289846.1). Importantly, we identified 34 potential recombination events that involved mostly strains from VII and Ib genotypes. A recombinant of genotype XII isolated in 2019 seems to emerge newly in Southern China. Further, the vaccine strains are found to be highly involved in potential recombination. Therefore, since the influence of recombination on NDV virulence cannot be predicted, this report's findings need to be considered for the security of NDV oncolytic application and the safety of NDV live attenuated vaccines.

Cell-Permeable Ubiquitin and Histone Tools for Studying Post-translational Modifications.

Protein post-translational modifications (PTMs) regulate nearly all biological processes in eukaryotic cells, and synthetic PTM protein tools are widely used to detect the activity of the related enzymes and identify the interacting proteins in cell lysates. Recently, the study of these enzymes and the interacting proteome has been accomplished in live cells using cell-permeable PTM protein tools. In this concept, we will introduce cell penetrating techniques, the syntheses of cell-permeable PTM protein tools, and offer some future perspective.

Multichromosomal Mitochondrial Genome of Paphiopedilum micranthum: Compact and Fragmented Genome, and Rampant Intracellular Gene Transfer.

Orchidaceae is one of the largest families of angiosperms. Considering the large number of species in this family and its symbiotic relationship with fungi, Orchidaceae provide an ideal model to study the evolution of plant mitogenomes. However, to date, there is only one draft mitochondrial genome of this family available. Here, we present a fully assembled and annotated sequence of the mitochondrial genome (mitogenome) of Paphiopedilum micranthum, a species with high economic and ornamental value. The mitogenome of P. micranthum was 447,368 bp in length and comprised 26 circular subgenomes ranging in size from 5973 bp to 32,281 bp. The genome encoded for 39 mitochondrial-origin, protein-coding genes; 16 tRNAs (three of plastome origin); three rRNAs; and 16 ORFs, while rpl10 and sdh3 were lost from the mitogenome. Moreover, interorganellar DNA transfer was identified in 14 of the 26 chromosomes. These plastid-derived DNA fragments represented 28.32% (46,273 bp) of the P. micranthum plastome, including 12 intact plastome origin genes. Remarkably, the mitogenome of P. micranthum and Gastrodia elata shared 18% (about 81 kb) of their mitochondrial DNA sequences. Additionally, we found a positive correlation between repeat length and recombination frequency. The mitogenome of P. micranthum had more compact and fragmented chromosomes compared to other species with multichromosomal structures. We suggest that repeat-mediated homologous recombination enables the dynamic structure of mitochondrial genomes in Orchidaceae.

Evolutionary adaptation of bovine coronavirus (BCoV): Screening of natural recombinations across the complete genomes.

Bovine coronavirus (BCoV) is a member of pathogenic Betacoronaviruses that has been circulating for several decades in multiple host species. Given the similarity between BCoV and human coronaviruses, the current study aimed to review the complete genomes of 107 BCoV strains available on the GenBank database, collected between 1983 and 2017 from different countries. The maximum-likelihood based phylogenetic analysis revealed three main BCoV genogroups: GI, GII, and GIII. GI is further divided into nine subgenogroups: GI-a to GI-i. The GI-a to GI-d are restricted to Japan, and GI-e to GI-i to the USA. The evolutionary relationships were also inferred using phylogenetic network analysis, revealing two major distinct networks dominated by viruses identified in the USA and Japan, respectively. The USA strains-dominated Network Cluster includes two sub-branches: France/Germany and Japan/China in addition to the United States, while Japan strains-dominated Network Cluster is limited to Japan. Twelve recombination events were determined, including 11 intragenogroup (GI) and one intergenogroup (GII vs. GI-g). The breakpoints of the recombination events were mainly located in ORF1ab and the spike glycoprotein ORF. Interestingly, 10 of 12 recombination events occurred between Japan strains, one between the USA strains, and one from intercontinental recombination (Japan vs. USA). These findings suggest that geographical characteristics, and population density with closer contact, might significantly impact the BCoV infection and co-infection and boost the emergence of more complex virus lineages.

α-Subunit Tyrosine Phosphorylation Is Required for Activation of the Large Conductance Ca2+-Activated Potassium Channel in the Rabbit Sphincter of Oddi.

Large conductance Ca2+-activated potassium (BKCa) channels are regulated by intracellular free Ca2+ concentrations ([Ca2+]i) and channel protein phosphorylation. In hypercholesterolemia (HC), motility impairment of the sphincter of Oddi (SO) is associated with abnormal [Ca2+]i accumulation in smooth muscle cells of the rabbit SO (RSOSMCs), which is closely related to BKCa channel activity. However, the underlying mechanisms regulating channel activity remain unclear. In this study, an HC rabbit model was generated and used to investigate BKCa channel activity of RSOSMCs via SO muscle tone measurement in vitro and manometry in vivo, electrophysiological recording, intracellular calcium measurement, and Western blot analyses. BKCa channel activity was decreased, which correlated with [Ca2+]i overload and reduced tyrosine phosphorylation of the BKCa α-subunit in the HC group. The abnormal [Ca2+]i accumulation and decreased BKCa channel activity were partially restored by Na3VO4 pretreatment but worsened by genistein in RSOSMCs in the HC group. This study suggests that α-subunit tyrosine phosphorylation is required for [Ca2+]i to activate BKCa channels, and there is a negative feedback between the BKCa channel and the L-type voltage-dependent Ca2+ channel that regulates [Ca2+]i. This study provides direct evidence that tyrosine phosphorylation of BKCa α-subunits is required for [Ca2+]i to activate BKCa channels in RSOSMCs, which may be the underlying physiological and pathologic mechanism regulating the activity of BKCa channels in SO cells.

Global Distribution and Natural Recombination of Hepatitis D Virus: Implication of Kyrgyzstan Emerging HDVs in the Clinical Outcomes.

Discrepancies in human hepatitis delta virus (HDV) genotypes impact the virus' biological behavior, clinical manifestation, and treatment response. Herein, this report aims to explore the role of recombination in the worldwide genotypic distribution and genetic diversity of HDV. Three-hundred-forty-eight human HDV full-length genomic sequences of ~1678 nt in length, isolated in twenty-eight countries worldwide between 1986 and 2018, were analysed. Similarity analysis and recombination mapping were performed, and forty-eight recombination events were identified, twenty-nine of which were isolated from Kyrgyzstan and determined to be involved in the diversity and extension of HDV sub-genotypes. HDV recombination occurred only between the genetically close genotypes (genotype 5 and genotype 2) or mainly within genotype 1, suggesting the complex replicative molecular mechanisms of HDV-RNA. The global distribution and classification of HDV genotypes have been updated, indicating that HDV recombination is one of the driving forces behind the biodiversity and the evolution of human HDV genomes. The outcome analysis suggests that the expansion of HDV sub-genotypes and the complex recombination networks might be related to the genomic character of Kyrgyzstan circulating strains and extensive mobility within countries and across borders. These findings will be of great importance in formulating more effective public health HDV surveillance strategies and guiding future molecular and epidemiological research to achieve better clinical outcomes.

Genomic characteristics and recombination patterns of swine hepatitis E virus in China.

Zoonotic hepatitis E, mainly caused by swine hepatitis E virus (sHEV), is endemic in China, causing great economic disruption and public health threats. Although recombination is critical for the evolution of viruses, there is a limited assessment of its occurrence among sHEVs. Herein, we analysed all available sHEV full-length genomes isolated in China during the past two decades (40 isolates) compared to 72 other sHEV strains isolated in different countries and determined that sHEV genotype 4 (sHEV4) dominates China. Eight potential natural recombination events were identified, four of which occurred in China and were mainly between sHEV4 strains, indicating the distinct character of China sHEV. One intergenotype recombination event was found in China, alarming the emergence of a new sHEV lineage that could become a critical threat to human health.

Genetic Characteristics of Porcine Hemagglutinating Encephalomyelitis Coronavirus: Identification of Naturally Occurring Mutations Between 1970 and 2015.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a Betacoronavirus characterized by neurological symptoms and a worldwide prevalence. Although PHEV is one of the earliest discovered porcine coronaviruses, it remains poorly studied. The full-length genome of the earliest PHEV strain collected in 1970 in the United States (PHEV/67 N/US/1970) was determined in October 2020. Using this virus as a prototype, we comparatively analyzed all available PHEV full-length sequences during 1970-2015. In phylogenetic trees based on PHEV full-length or spike glycoprotein open reading frame genomic sequences, PHEV/67 N/US/1970 was sorted into a clade different from that of viruses isolated in the United States in 2015. Intriguingly, United States and Belgium viruses isolated in 2015 and 2005, respectively, revealed multiple deletion mutation patterns compared to the strain PHEV/67 N/US/1970, leading to a truncated or a non-functional NS2A coding region. In addition, the genomic similarity analysis showed a hypervariability of the spike glycoprotein coding region, which can affect at least eight potential linear B cell epitopes located in the spike glycoprotein. This report indicates that PHEVs in the United States underwent a significant genetic drift, which might influence PHEV surveillance in other countries.

Corrigendum: Expression of Pumpkin CmbHLH87 Gene Improves Powdery Mildew Resistance in Tobacco.

[This corrects the article DOI: 10.3389/fpls.2020.00163.].

The Characteristics of Spike Glycoprotein Gene of Swine Acute Diarrhea Syndrome Coronavirus Strain CH/FJWT/2018 Isolated in China.

Swine acute diarrhea syndrome (SADS) is a highly contagious infectious disease characterized by acute vomiting and watery diarrhea in neonatal piglets. The causative agent for SADS is the swine acute diarrhea syndrome coronavirus (SADS-CoV), an alphacoronavirus in the family Coronaviridae. Currently, SADS-CoV was identified only in Guangdong and Fujian provinces of China, not in any other regions or countries in the world. To explore the genetic diversity of SADS-CoV isolates, herein we comparatively analyzed 44 full-length genomes of viruses isolated in Guangdong and Fujian provinces during 2017-2019. The spike glycoprotein gene of SADS-CoV strain CH/FJWT/2018 isolated in Fujian province is distinct from that of other viral isolates in either spike glycoprotein gene-based phylogenetic analysis or whole genome-based gene similarity analysis. Moreover, at least 7 predicted linear B cell epitopes in the spike glycoprotein of CH/FJWT/2018 would be affected by amino acid variations when compared with a representative virus isolated in Guangdong province. The spike glycoprotein of coronaviruses determines viral host range and tissue tropism during virus infection via specific interactions with the cellular receptor and also plays critical roles in eliciting the production of neutralizing antibodies. Since SADS-CoVs have a broad cell tropism, the results in this report further emphasize that the spike glycoprotein gene is a pivotal target in the surveillance of SADS-CoV.

Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS.

Filamentous fungi identification by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenging due to the lack of simple and rapid protein extraction methods and insufficient species coverage in the database. In this study, we created two rapid protein extraction methods for filamentous fungi: a one-step zirconia-silica beads method (ZSB) and a focused-ultrasonication method (FUS). The identification accuracy of two methods were evaluated with the VITEK MS, as well as number of spectra peaks and signal-to-noise ratio (S/N) with M-Discover 100 MALDI-TOF MS compared to the routine method. The better method was applied to build a filamentous fungi in-house spectra library for the M-Discover 100 MS, and then another one and routine method were performed in parallel to verify the accuracy and commonality of the in-house library. Using the two optimized methods, the dedicated operating time before MALDI-TOF MS analysis was reduced from 30 min to 7 (ZSB) or 5 (FUS) min per sample, with only a few seconds added for each additional strain. And both two methods identified isolates from most mold types equal to or better than the routine method, and the total correct identification rate using VITEK MS was 79.67, 76.42, and 76.42%, respectively. On the other hand, the two rapid methods generally achieved higher maximum and minimum S/N ratios with these isolates tested as compared to the routine method. Besides, the ZSB method produced overall mean of maximum and minimum S/N ratio higher than that by FUS. An in-house library of M-Discover MS was successfully built from 135 isolates from 42 species belonging to 18 genera using the ZSB method. Analysis of 467 isolates resulted in 97.22% correctly identified isolates to the species level by the ZSB method versus 95.50% by the routine method. The two novel methods are time- and cost-effective and allow efficient identification of filamentous fungi while providing a simplified procedure to build an in-house library. Thus, more clinical laboratories may consider adopting MALDI-TOF MS for filamentous fungi identification in the future.