Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.

N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.

The efficacy and safety of acupoint catgut embedding for peripheral facial paralysis sequela: A protocol for systematic review and meta-analysis.

BACKGROUND: Peripheral facial paralysis sequela (PFPS) is a group of sequence syndrome after the acute onset of peripheral facial paralysis. Nearly 70% of patients with peripheral facial paralysis recover completely, but nearly 30% of patients leave multiple sequelae, which have serious negative impacts on the physical and psychological health of patients. Without a high risk of side effect, acupoint catgut embedding (ACE), a common acupuncture therapy, is widely used to treat this disorder. And a number of studies have shown the efficacy of this therapy for PFPS. But in fact, the evidence of the overall effect of ACE in the treatment of PFPS is still insufficient. Therefore, the purpose of this study is to evaluate the efficiency and safety of ACE for PFPS. METHODS: Two reviewers will collect randomized controlled trials (RCTs) on ACE for PFPS by searching the following databases, including The Cochrane Library, PubMed, Web of Science, EMBASE, China Biomedical Literature (CBM), China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), and Wanfang database, from their initiation to May 2021. The searching of publications will include English and Chinese without any restriction of countries and regions. Besides, 2 reviewers will independently include in studies that meet the inclusion criteria and extract data we need, then use Cochrane Collaboration's Risk of Bias Tool to assess their methodological quality. The efficacy and safety of ACE as a treatment for PFPS will be assessed according to the synthetic risk ratio (RR), odds ratio (OR), or weighted mean difference (WMD), standardized mean difference (SMD) with consistent 95% confidence intervals (95% CI). And the Review Manager 5.3 software will be adopted to conduct the statistical analysis. RESULTS: The protocol for meta-analysis will systematically evaluate the efficacy and safety of ACE for PFPS. And the final result of this search will provide sufficient evidence and an authentic assessment focusing on the problem. CONCLUSION: This search will explore whether ACE could be used as an effective and non-drug external therapy of TCM for PFPS and offer supports for clinical practice. PROSPERO REGISTRATION NUMBER: CRD42021240004.

Starch gelatinization in the surface layer of rice grains is crucial in reducing the stickiness of parboiled rice.

Parboiled rice has high nutritional value but unpleasant palatability. In this study, rice stickiness was significantly reduced by steaming during the parboiling process; however, continuing steaming past certain durations no longer affected rice stickiness. It was also found: (i) the degree of starch gelatinization (DSG) increases and starch crystallinity decreases with increasing steaming time; (ii) the molecular size and chain length distribution (CLD) of leached starch for both white and parboiled rice are significantly different from those of native starch; (iii) the relation between leached amylopectin amount and rice stickiness explains the reduced stickiness by parboiling; and (iv) starch gelatinization in the surface layer of rice grains during parboiling might be critically important in blocking starch leaching, consequently leading to a less sticky texture. This study supplies a way to manage glutinous rice stickiness by parboiling for the production of non-sticky rice foods.

Structural and physicochemical property changes during pyroconversion of native maize starch.

Pyrodextrins are widely applied in industry as adhesives, coatings, binders, and in food as dietary fiber. In this study, the dynamic changes of structure and physicochemical property during pyroconversion of native maize starch are investigated. By quantifying the variations of chain-length distribution (CLD) between native starch and pyrodextrins, it is found that 1) the long starch chains with degree of polymerization (DP) about 500-20000 are degraded while its content is not significantly varied between pyrodextrins; 2) the content of intermediate-long chains with DP 20-500 increases with temperature, indicating more pyrodextrin molecules with the equivalent Rh∼1.5-9.4 nm are newly formed; 3) the content of A and B1 chains are not largely varied between pyrodextrins with temperature ≤190 °C, suggesting that the repolymerization might be more likely occur in the amorphous lamellae. This study improves the understanding of structural and physicochemical property changes during the pyroconversion process of native maize starch.

Insights into maize starch degradation by high pressure homogenization treatment from molecular structure aspect.

High-pressure homogenization (HPH) is a common physical method used for starch modification. In this study, starch molecular structure in terms of chain-length distribution (CLD) and molecular size is characterized to explore the structural variations during HPH and its internal relations. It is found that: 1) the molecular size is significantly reduced by HPH treatments and further gradually decreases with HPH pressure increasing; 2) HPH degrades the long amylose chains with degree of polymerization (DP) ~ 2000-20,000 into short- and intermediate-amylose chains with DP ~ 100-1000 and DP ~ 1000-2000; 3) by HPH treatment, the proportion of amylopectin chains with DP ~ 6-12 and DP ~ 12-24 decreases while that with DP ~ 24-36 and DP ~ 36-100 increases, whereas, the amylopectin CLDs between HPH treated starch samples are not significantly varied; and 4) by a subtraction analysis, the molecular size of HPH treated starches shows a strong correlation with the proportion of degraded long amylose chains, indicating these long amylose chains might play a critical role in maintaining the large molecular size of starch. This study provides a further understanding of molecular features from the individual chains assembling into a whole branched molecule.

Effect of dry heating treatment on multi-levels of structure and physicochemical properties of maize starch: A thermodynamic study.

Dry heating treatment (DHT) is a common process widely used in food industry. In this study, the thermodynamic effects of DHT on starch structure and physicochemical properties are investigated. The results show that, with heating temperature increasing during DHT, the molecular size, long-amylose chains with degree of polymerization (DP) ~5000-20,000, and the crystallinity of maize starch are significantly reduced while the granular structure is retained with slightly aggregation between starch particles. The solubility of DHT starch increases from 0.5% to 2%, indicating the majority of DHT starch is still insoluble. DHT affects starch thermal property greatly that, it decreases the gelatinization enthalpy while increases the heterogeneity of starch crystallites. With heating temperature increasing, DHT reduces the overall viscosity of starch paste. The rheological property of DHT starch is frequency-dependent, showing the typical shear thinning behavior and "solid-like" gel property. Especially, as heating temperature reaches 190 °C, the shear resistance becomes stronger, and it is closer to Newtonian fluid. The results prove the thermodynamic effects of DHT on multi-levels of starch structure and physicochemical properties, also indicating the great potential to utilize DHT in modifying starch properties and amplifying its applications.

Insights into maize starch degradation by sulfuric acid from molecular structure changes.

In this study, the molecular structure of hydrolyzed maize starch by sulfuric acid is analyzed by characterizing the chain-length distribution (CLD) and molecular size. It is found that molecular size is dramatically reduced by acid hydrolysis for only 1 h while amylose content is gradually decreased from 28.3 % to 4.6 % with hydrolysis time prolonging. By quantifying the subtraction of CLDs between hydrolyzed sample and native starch, we find: 1) the long chains with degree of polymerization (DP)∼500-30000 of native starch are degraded by acid hydrolysis for only 1 h; 2) with hydrolysis time extending, the degradation of short-amylose chains and even short amylopectin chains are initialized; 3) molecular size of hydrolyzed starch shows a strong correlation with the parameters derived from the subtraction analysis. This study could help to further understand molecular features from the individual chain assembling to a fully branched molecule.

The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development.

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism.

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System.

Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

MTV, an ssDNA Protecting Complex Essential for Transposon-Based Telomere Maintenance in Drosophila.

Multiple complexes protect telomeres. In telomerase-maintained organisms, Shelterin related complexes occupy the duplex region while the CST and Tpp1-Pot1 complexes bind the single stranded overhang of telomeres. Drosophila uses a transposon-based mechanism for end protection. We showed that the HOAP-HipHop complex occupies the duplex region. Whether an ssDNA-binding complex exists is not known. Here we discover a novel protein, Tea, that is specifically enriched at telomeres to prevent telomere fusion. We also identify a complex consisting of Tea and two known capping proteins, Ver and Moi. The Moi-Tea-Ver (MTV) complex purified in vitro binds and protects ssDNA in a sequence-independent manner. Tea recruits Ver and Moi to telomeres, and point mutations disrupting MTV interaction in vitro result in telomere uncapping, consistent with these proteins functioning as a complex in vivo. MTV thus shares functional similarities with CST or TPP1-POT1 in protecting ssDNA, highlighting a conserved feature in end protecting mechanisms.

Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System.

Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in antibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in response to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type-specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-α/ß receptor-mediated feed-forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation.