High homocysteine levels as potential indicators of subacute combined degeneration of the spinal cord.

BACKGROUND: Homocysteine (Hcy) is widely recognized as a significant risk factor for cardiovascular and cerebrovascular diseases. However, our research has uncovered a novel perspective, suggesting that elevated levels of Hcy could serve as an indicator for neurological diseases. This article presents a unique case of Subacute Combined Degeneration of the spinal cord(SCD), characterized by high homocysteine levels, yet normal vitamin B12 and imaging results. This discovery could facilitate early detection and ensure timely referral of patients to specialized departments for further treatment.

Multiple Roles of the Low-Affinity Calcium Uptake System in Drechslerella dactyloides, a Nematode-Trapping Fungus That Forms Constricting Rings.

(1) Background: the low-affinity calcium uptake system (LACS) has been shown to play a crucial role in the conidiation and formation of adhesive nets and knobs by nematode-trapping fungi (NTF), but its involvement in the formation of constricting rings (CRs), mechanical traps to capture free-living nematodes, remains unexplored. (2) Methods: we investigated the function of two LACS genes (DdaFIG_1 and DdaFIG_2) in Drechslerella dactyloides, an NTF that forms CRs. We generated single (DdaFIG_1Ri and DdaFIG_2Ri) and double (DdaFIG_1,2Ri) knockdown mutants via the use of RNA interference (RNAi). (3) Results: suppression of these genes significantly affected conidiation, trap formation, vegetative growth, and response to diverse abiotic stresses. The number of CRs formed by DdaFIG_1Ri, DdaFIG_2Ri, and DdaFIG_1,2Ri decreased to 58.5%, 59.1%, and 38.9% of the wild-type (WT) level, respectively. The ring cell inflation rate also decreased to 73.6%, 60.6%, and 48.8% of the WT level, respectively. (4) Conclusions: the LACS plays multiple critical roles in diverse NTF.

Tools and basic procedures of gene manipulation in nematode-trapping fungi.

Nematode-trapping fungi (NTF) are the majority of carnivorous microbes to capture nematodes through diverse and sophisticated trapping organs derived from hyphae. They can adopt carnivorous lifestyles in addition to saprophytism to obtain extra-nutrition from nematodes. As a special group of fungi, the NTF are not only excellent model organism for studying lifestyle transition of fungi but also natural resources of exploring biological control of nematodes. However, the carnivorous mechanism of NTF remains poorly understood. Nowadays, the omics studies of NTF have provided numerous genes and pathways that are associated with the phenotypes of carnivorous traits, which need molecular tools to verify. Here, we review the development and progress of gene manipulation tools in NTF, including methodology and strategy of transformation, random gene mutagenesis methods and target gene mutagenesis methods. The principle and practical approach for each method was summarized and discussed, and the basic operational flow for each tool was described. This paper offers a clear reference and instruction for researchers who work on NTF as well as other group of fungi.

DhFIG_2, a gene of nematode-trapping fungus Dactylellina haptotyla that encodes a component of the low-affinity calcium uptake system, is required for conidiation and knob-trap formation.

Calcium ion (Ca2+) is a universal second messenger involved in regulating diverse processes in animals, plants, and fungi. The low-affinity calcium uptake system (LACS) participates in acquiring Ca2+ from extracellular environments under high extracellular Ca2+ concentration. Unlike most fungi, which encode only one protein (FIG1) for LACS, nematode-trapping fungi (NTF) encode two related proteins. AoFIG_2, the NTF-specific LACS component encoded by adhesive network-trap forming Arthrobotrys oligospora, was shown to be required for conidiation and trap formation. We characterized the role of DhFIG_2, an AoFIG_2 ortholog encoded by knob-trap forming Dactylellina haptotyla, in growth and development to expand our understanding of the role of LACS in NTF. Because repeated attempts to disrupt DhFIG_2 failed, knocking down the expression of DhFIG_2 via RNA interference (RNAi) was used to study its function. RNAi of DhFIG_2 significantly decreased its expression, severely reduced conidiation and trap formation, and affected vegetative growth and stress responses, suggesting that this component of LACS is crucial for trap formation and conidiation in NTF. Our study demonstrated the utility of RNAi assisted by ATMT for studying gene function in D. haptotyla.

Neuroprotective Role of MiRNA-9 in Neurological Diseases: A Mini Review.

MicroRNAs (miRNAs) are a class of non-coding small RNAs with about 22 nucleotides in eukaryotes. They regulate gene expression at the post-transcriptional level and play a key role in physiological and pathological processes. As one of the most abundant miRNAs in the human brain, miRNA-9 (miR-9) has attracted extensive attention due to its important role in the maintenance of normal function of the nervous system and the occurrence and development of nervous system diseases. Hence, we reviewed the neuroprotective effect of miR-9 in neurological diseases. MiR-9 may be a potential target of nervous system diseases.

DdaCrz1, a C2H2-Type Transcription Factor, Regulates Growth, Conidiation, and Stress Resistance in the Nematode-Trapping Fungus Drechslerella dactyloides.

The Ca2+/calmodulin-dependent signaling pathway regulates diverse cellular processes. Calcineurin is a calcium-dependent phosphatase acting in fungi mainly through Crz1, a zinc finger transcription factor. Although the likely involvement of Ca2+ in fungal carnivorism has been documented, how Crz1 functions in nematode-trapping fungi remains unknown. Here, we identified the Crz1 gene (named as DdaCrz1) in Drechslerella dactyloides, a species that forms constricting rings to trap nematodes. The deletion of DdaCrz1 significantly reduced hyphal growth and conidiation, trap formation, and ring cell inflation. Moreover, the mutation increased sensitivity to Mn2+ but decreased sensitivity to Ca2+, Mg2+, Zn2+, and Li+. Similarly, the mutant showed increased tolerance to osmotic stress but was more sensitive to Congo red, a cell wall-damaging agent. Our results confirmed the critical roles of the Ca2+/calmodulin-dependent signaling pathway in regulating growth, conidiation, and the stress response, and suggested its involvement in trapping nematodes.

The Role of Glyceraldehyde-3-Phosphate Dehydrogenases in NADPH Supply in the Oleaginous Filamentous Fungus Mortierella alpina.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme within the glycolytic pathway. GAPDH catalyzes the transformation of glyceraldehyde 3-phosphate to glycerate-1, 3-biphosphate, a process accompanied by the production of NADH. Its role in the NADPH production system of the oleaginous filamentous fungus Mortierella alpina was explored. Two copies of genes encoding GAPDH were characterized, then endogenously overexpressed and silenced through Agrobacterium tumefaciens-mediated transformation methods. The results showed that the lipid content of the overexpression strain, MA-GAPDH1, increased by around 13%. RNA interference of GAPDH1 and GAPDH2 (MA-RGAPDH1 and MA-RGAPDH2) greatly reduced the biomass of the fungus. The lipid content of MA-RGAPDH2 was found to be about 23% higher than that of the control. Both of the lipid-increasing transformants showed a higher NADPH/NADP ratio. Analysis of metabolite and enzyme expression levels revealed that the increased lipid content of MA-GAPDH1 was due to enhanced flux of glyceraldehyde-3-phosphate to glycerate-1, 3-biphosphate. MA-RGAPDH2 was found to strengthen the metabolic flux of dihydroxyacetone phosphate to glycerol-3-phosphate. Thus, GAPDH1 contributes to NADPH supply and lipid accumulation in M. alpina, and has a distinct role from GAPDH2.

An efficient strategy for screening polyunsaturated fatty acid-producing oleaginous filamentous fungi from soil.

Screening oleaginous microorganisms capable of accumulating considerable lipids is essential for industrial lipid production. Here we demonstrated forty-seven filamentous fungal isolates were obtained from eight soil samples using a new screening strategy with both triphenyltetrazolium chloride (TTC), a redox indicator used for testing oil presence, and cerulenin, an inhibitor of fatty acid synthase (FAS), supplemented in screening medium. Among these fungal isolates, nineteen have high lipid content (>20% dry biomass weight) and were affiliated with the genus Mortierella by morphology identification and phylogenetic analysis based on ITS gene sequences. Notably, one strain designated as SL-4 reached 32% of its biomass weight as lipid, displaying the highest potential. Two candidates with high lipid content as well as biomass production were selected for exploring the effect of different carbon and nitrogen sources on morphology, biomass and lipid accumulation.

Optimization of Agrobacterium tumefaciens-mediated transformation method of oleaginous filamentous fungus Mortierella alpina on co-cultivation materials choice.

The Agrobacterium tumefaciens-mediated transformation (ATMT) method is commonly applied in the oleaginous filamentous fungus Mortierella alpina. During the ATMT process, the spores of M. alpina have traditionally been used as a co-cultivation material, but their long spore-producing cycle and low sporulation rate make the transformation process tedious. This study explores the use of germinating spores, mycelium and single solid colonies of uracil auxotrophic M. alpina CCFM501 as a co-cultivation material with A. tumefaciens AGL1. The results show that A. tumefaciens AGL1 can successfully transform the germinating spores, mycelium and single solid colonies of M. alpina. In addition, the transformation rate of the germinating spores was 50% higher than that of the fresh spores. Due to its concise preparation process, the mycelium was chosen as a co-cultivation material for two plasmids of different lengths and proven to be an efficient co-cultivation material for M. alpina.

Molecular tools for gene manipulation in filamentous fungi.

Functional genomics of filamentous fungi has gradually uncovered gene information for constructing 'cell factories' and controlling pathogens. Available gene manipulation methods of filamentous fungi include random integration methods, gene targeting technology, gene editing with artificial nucleases and RNA technology. This review describes random gene integration constructed by restriction enzyme-mediated integration (REMI); Agrobacterium-mediated transformation (AMT); transposon-arrayed gene knockout (TAGKO); gene targeting technology, mainly about homologous recombination; and modern gene editing strategies containing transcription activator-like effector nucleases (TALENs) and a clustered regularly interspaced short palindromic repeat/associated protein system (CRISPR/Cas) developed in filamentous fungi and RNA technology including RNA interference (RNAi) and ribozymes. This review describes historical and modern gene manipulation methods in filamentous fungi and presents the molecular tools available to researchers investigating filamentous fungi. The biggest difference of this review from the previous ones is the addition of successful application and details of the promising gene editing tool CRISPR/Cas9 system in filamentous fungi.

IL-17F but not IL-17A gene polymorphism confers risk to multiple sclerosis in a Chinese Han population.

Interleukin-17 has been shown to be associated with autoimmune disease. The aim of the current study is to investigate the potential association of IL-17 polymorphisms with multiple sclerosis (MS) in Chinese Han patients. Two SNPs, rs763780 of IL-17F gene and rs2275913 of IL-17A gene were genotyped in 622 MS patients and 743 healthy controls by using a polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Allele and genotype frequencies distribution of the two SNPs were examined between patients and controls using the Chi-Square test. All genotypic and allelic frequencies of the tested IL-17 polymorphisms in control cohort were in Hardy-Weinberg equilibrium. A significantly increased frequency of rs763780 TT genotype (corrected p value (Pc)=0.024, odds ratio=1.472, 95% CI=1.133-1.913) and T allele (corrected P (Pc)=0.018, odds ratio=1.446, 95% CI=1.134-1.844) was detected in MS patients compared with controls. The genotypic and allelic frequencies of rs2275913 in IL-17A gene were not different between patients with MS and controls. These results suggest that rs763780 is associated with multiple sclerosis in a Chinese Han population.

Genetic polymorphisms in pre-microRNAs and risk of ischemic stroke in a Chinese population.

Ischemic stroke is considered to be a complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors. MicroRNAs participated in various physiopathological processes; common single-nucleotide polymorphisms (SNPs) in pre-miRNAs have been shown to be associated with susceptibility to several human diseases. We evaluated the associations of the hsa-mir-196-a2/rs11614913 T/C, hsa-mir-146a/rs2910164 C/G, and hsa-mir-499/rs3746444 A/G polymorphisms in pre-miRNAs with the risk of ischemic stroke in a Chinese population. The three polymorphisms were identified in 296 ischemic stroke patients and 391 healthy controls using polymerase chain reaction-restriction fragment length polymorphism. The frequency of the allele G of hsa-mir-499/rs3746444 A/G showed significant association with ischemic stroke when compared with controls (OR = 1.509, 95%CI = 1.151-1.978, P = 0.003). Increased ischemic stroke risks were associated with rs3746444 A/G genotypes in different genetic model (homozygote comparison: P = 0.045, OR = 2.084, 95%CI = 1.019-4.262; heterozygote comparison: P = 0.024, OR = 1.489, 95%CI = 1.063-2.087; dominant genetic model: P = 0.007, OR = 1.563, 95%CI = 1.135-2.153). Similar results were obtained by adjusted fully risk factors. However, we failed to find any association between the alleles and genotypes of rs2910164 C/G and rs11614913 T/C SNPs and ischemic stroke, respectively (p > 0.05). The present study provided evidence that hsa-mir-499/rs3746444 A/G polymorphism might be associated with a significantly increased risk of ischemic stroke in a Chinese population, indicating that the common genetic polymorphism in pre-microRNAs contributed to the pathogenesis of ischemic stroke.

Single nucleotide polymorphism of CD40 in the 5'-untranslated region is associated with ischemic stroke.

OBJECTIVES: Ischemic stroke is influenced by both environmental and genetic factors. The CD40/CD40L system is related to proinflammatory and prothrombogenic responses, which are involved in the pathophysiology of ischemic stroke. The aim of this study was to evaluate association between the CD40 -1C/T single nucleotide polymorphism (SNP) and ischemic stroke in a Chinese population. METHODS: We conducted a case-control study including 286 ischemic stroke patients and 336 controls. CD40 -1C/T SNP was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods, and evaluated its relevance to ischemic stroke susceptibility. RESULTS: Significantly increased ischemic stroke risk was found to be associated with the T allele of CD40 -1C/T (OR=1.273, 95% CI=1.016-1.594). The frequencies of CT and TT/CT genotypes of CD40 -1C/T in ischemic stroke patients were significantly higher than those of controls, respectively (for CT: OR=2.350, 95% CI=1.601-3.449; for TT/CT: OR=2.148, 95% CI=1.479-3.119). And, similar results were obtained after adjusting non-matched variables. We found that the frequency of carried T genotypes (TT and TT/CT) was significantly increased in patients with history of stroke compared with patients without (for TT: OR=6.538, 95%CI=1.655-25.833; for TT/CT: OR=3.469, 95%CI=1.031-11.670), respectively. CONCLUSIONS: The findings suggested that the CD40 -1C/T polymorphism might contribute to the susceptibility to ischemic stroke in the Chinese population, and might be associated with history of previous stroke.