Pseudoexon activation by deep intronic variation in GNE myopathy with thrombocytopenia.

INTRODUCTION/AIMS: GNE myopathy is a rare autosomal recessive disorder caused by pathogenic variants in the GNE gene, which is essential for the sialic acid biosynthesis pathway. Although over 300 GNE variants have been reported, some patients remain undiagnosed with monoallelic pathogenic variants. This study aims to analyze the entire GNE genomic region to identify novel pathogenic variants. METHODS: Patients with clinically compatible GNE myopathy and monoallelic pathogenic variants in the GNE gene were enrolled. The other GNE pathogenic variant was verified using comprehensive methods including exon 2 quantitative polymerase chain reaction and nanopore long-read single-molecule sequencing (LRS). RESULTS: A deep intronic GNE variant, c.862+870C>T, was identified in nine patients from eight unrelated families. This variant generates a cryptic splice site, resulting in the activation of a novel pseudoexon between exons 5 and 6. It results in the insertion of an extra 146 nucleotides into the messengerRNA (mRNA), which is predicted to result in a truncated humanGNE1(hGNE1) protein. Peanut agglutinin（PNA） lectin staining of muscle tissues showed reduced sialylation of mucin O-glycans on sarcolemmal glycoproteins. Notably, a third of patients with the c.862+870C>T variant exhibited thrombocytopenia. A common core haplotype harboring the deep intronic GNE variant was found in all these patients. DISCUSSION: The transcript with pseudoexon activation potentially affects sialic acid biosynthesis via nonsense-mediated mRNA decay, or resulting in a truncated hGNE1 protein, which interferes with normal enzyme function. LRS is expected to be more frequently incorporated in genetic analysis given its efficacy in detecting hard-to-find pathogenic variants.

Cellular and Molecular Mechanisms Underlying Synaptic Subcellular Specificity.

Chemical synapses are essential for neuronal information storage and relay. The synaptic signal received or sent from spatially distinct subcellular compartments often generates different outcomes due to the distance or physical property difference. Therefore, the final output of postsynaptic neurons is determined not only by the type and intensity of synaptic inputs but also by the synaptic subcellular location. How synaptic subcellular specificity is determined has long been the focus of study in the neurodevelopment field. Genetic studies from invertebrates such as Caenorhabditis elegans (C. elegans) have uncovered important molecular and cellular mechanisms required for subcellular specificity. Interestingly, similar molecular mechanisms were found in the mammalian cerebellum, hippocampus, and cerebral cortex. This review summarizes the comprehensive advances in the cellular and molecular mechanisms underlying synaptic subcellular specificity, focusing on studies from C. elegans and rodents.

Genome assembly and annotation of the king ratsnake, Elaphe carinata.

The king ratsnake (Elaphe carinata) of the genus Elaphe is a common large, non-venomous snake widely distributed in Southeast and East Asia. It is an economically important farmed species. As a non-venomous snake, the king ratsnake predates venomous snakes, such as cobras and pit vipers. However, the immune and digestive mechanisms of the king ratsnake remain unclear. Despite their economic and research importance, we lack genomic resources that would benefit toxicology, phylogeography, and immunogenetics studies. Here, we used single-tube long fragment read sequencing to generate the first contiguous genome of a king ratsnake from Huangshan City, Anhui province, China. The genome size is 1.56 GB with a scaffold N50 of 6.53M. The total length of the genome is approximately 621 Mb, while the repeat content is 42.26%. Additionally, we predicted 22,339 protein-coding genes, including 22,065 with functional annotations. Our genome is a potentially useful addition to those available for snakes.

Prediction model of water content in surface dead fuel based on convolution neural network and meteoro-logical factors regression.

Water content of surface fuels is an important indicator of forest fire risk level and fire behavior, and the prediction model of which has a significant effect on fire risk prediction and management. Based on field meteorological factors of Quercus mongolica and Pinus sylvestris var. mongolica forests and water content data of dead fuels on the ground, we conducted the relative importance ranking of meteorological factors random forest and Pearson correlation analysis, and predicted water content of fuels using deeply learned convolutional neural network and meteorological factors regression. The results showed that water content of Q. mongolica fuels in the wild was significantly higher than that of P. sylvestris var. mongolica. The results of random forest showed that the factors that had significant effect on water content of fuel were humidity, temperature, rainfall, wind speed, and solar radiation, with the importance ranking from the largest to the smallest. Results of correlation analysis showed that temperature, humidity, and rainfall of current day had a significant impact on water content of fuels, and certain correlations were observed between meteorological factors. The prediction R2 of the convolutional neural network model for the surface fuel water content of Q. mongolica and P. sylvestris var. mongolica forest was 0.928 and 0.905, the mean absolute error (MAE) was 6.1% and 8.1%, and the mean relative error (MRE) was 8.9% and 4.2%, respectively. However, the R2, MAE, MRE of meteorological factors regression were 0.495 and 0.525, 30.5% and 39.5%, 52.1% and 32.6%, respectively. The precision of convolution neural network model was significantly higher than that of meteorological factors regression. Our results showed that the deeply learned convolutional neural network could provide some reference for the prediction of fuel water content in the future, and effectively support higher level forest fire management.

Prediction models of fire spread rate of Pinus koraiensis plantation's surface fuel.

Pinus koraiensis plantation has high fire risks due to high oil content in branches and leaves. The spread of surface fire is the main way of forest fire expansion. Understanding the surface fire spread rate can provide scientific guidance for fire fighting. We carried out a laboratory experiment with surface fuel of Pinus koraiensis plantation in Maoershan area of Heilongjiang Province. We set different levels of fuel moisture contents (5%, 15%, 25%), fuel loads (0.5, 0.7, 0.9, 1.1 kg·m-2), and slope (0°, 10°, 20°, 30°, 40°) to simulate the characteristics of fuel bed in the field, and quantified the spread rate by thermocouple method. We further compared and analyzed the prediction accuracy of Rothermel model, modified Rothermel model and random forest model, and evaluated the optimal model for predicting the surface fire spread rate of P. koraiensis plantation. The results showed that the overall efficacy of directly using the Rothermel model to predict the surface fire spread rate of P. koraiensis plantations was good, but the prediction result of the spread rate under the conditions of high slope and high moisture content was not satisfied. The Rothermel model after refitting the slope parameters and the random forest model had good prediction efficacy and similar prediction accuracy. The random forest model needed to be further evaluated and verified due to its own characteristics. The modified Rothermel model was more suitable for predicting the surface fire spread rate of P. koraiensis plantations at a slope range of 0°-40° than the others.

Transmission and Drug Resistance Genotype of Multidrug-Resistant or Rifampicin-Resistant Mycobacterium tuberculosis in Chongqing, China.

Multidrug-resistant or rifampicin-resistant tuberculosis (MDR/RR-TB) is a global barrier for the Stop TB plan. To identify risk factors for treatment outcome and cluster transmission of MDR/RR-TB, whole-genome sequencing (WGS) data of isolates from patients of the Chongqing Tuberculosis Control Institute were used for phylogenetic classifications, resistance predictions, and cluster analysis. A total of 223 MDR/RR-TB cases were recorded between 1 January 2018 and 31 December 2020. Elderly patients and those with lung cavitation are at increased risk of death due to MDR/RR-TB. A total of 187 MDR/RR strains were obtained from WGS data; 152 were classified as lineage 2 strains. Eighty (42.8%) strains differing by a distance of 12 or fewer single nucleotide polymorphisms were classified as 20 genomic clusters, indicating recent transmission. Patients infected with lineage 2 strains or those with occupations listed as "other" are significantly associated with a transmission cluster of MDR/RR-TB. Analysis of resistant mutations against first-line tuberculosis drugs found that 76 (95.0%) of all 80 strains had the same mutations within each cluster. A total of 55.0% (44 of 80) of the MDR/RR-TB strains accumulated additional drug resistance mutations along the transmission chain, especially against fluoroquinolones (63.6% [28 of 44]). Recent transmission of MDR/RR strains is driving the MDR/RR-TB epidemics, leading to the accumulation of more serious resistance along the transmission chains. IMPORTANCE The drug resistance molecular characteristics of MDR/RR-TB were elucidated by genome-wide analysis, and risk factors for death by MDR/RR-TB were identified in combination with patient information. Cluster characteristics of MDR/RR-TB in the region were analyzed by genome-wide analysis, and risk factors for cluster transmission (recent transmission) were analyzed. These analyses provide reference for the prevention and treatment of MDR/RR-TB in Chongqing.

Bromate reduction by Shewanella oneidensis MR-1 is mediated by dimethylsulfoxide reductase.

Microbial bromate reduction plays an important role in remediating bromate-contaminated waters as well as biogeochemical cycling of bromine. However, little is known about the molecular mechanism of microbial bromate reduction so far. Since the model strain Shewanella oneidensis MR-1 is capable of reducing a variety of oxyanions such as iodate, which has a high similarity to bromate, we hypothesize that S. oneidensis MR-1 can reduce bromate. Here, we conducted an experiment to investigate whether S. oneidensis MR-1 can reduce bromate, and report bromate reduction mediated by a dimethylsulfoxide reductase encoded with dmsA. S. oneidensis MR-1 is not a bromate-respiring bacterium but can reduce bromate to bromide under microaerobic conditions. When exposed to 0.15, 0.2, 0.25, 0.5, and 1 mM bromate, S. oneidensis MR-1 reduced bromate by around 100, 75, 64, 48, and 23%, respectively, within 12 h. In vivo evidence from gene deletion mutants and complemented strains of S. oneidensis MR-1 indicates that MtrB, MtrC, CymA, GspD, and DmsA are involved in bromate reduction, but not NapA, FccA, or SYE4. Based on our results as well as previous findings, a proposed molecular mechanism for bromate reduction is presented in this study. Moreover, a genomic survey indicates that 9 of the other 56 reported Shewanella species encode proteins highly homologous to CymA, GspD, and DmsA of S. oneidensis MR-1 by sequence alignment. The results of this study contribute to understanding a pathway for microbial bromate reduction.

Transcriptome analysis provides new insights into the tolerance and aerobic reduction of Shewanella decolorationis Ni1-3 to bromate.

As a possible human carcinogen, bromate is easily formed in drinking water and wastewater treatments using advanced oxidation technology. Microbial reduction is a promising method to remove bromate, but little is known about aerobic bromate reduction as well as the molecular mechanism of tolerance and reduction to bromate in bacteria. Herein, bromate reduction by isolate under aerobic conditions was reported for the first time. Shewanella decolorationis Ni1-3, isolated from an activated sludge recently, was identified to reduce bromate to bromide under both aerobic and anaerobic conditions. RNA-Seq together with differential gene expression analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify that bromate triggered the expression of genes for oxidative stress protection (e.g., ohr, msrQ, dsbC, gpo, gorA, and gst), DNA damage repair (e.g., dprA, parA, and recJ), and sulfur metabolism (e.g., cysH, cysK, and cysP). However, the genes for lactate utilization (e.g., lldF and dld), nitrate reduction (e.g., napA and narG), and dissimilatory metal reduction (e.g., mtrC and omcA) were down-regulated in the presence of bromate. The results contribute to revealing the molecular mechanism of resistance and reduction in S. decolorationis Ni1-3 to bromate under aerobic conditions and clarifying the biogeochemical cycle of bromine. KEY POINTS: • Aerobic bromate reduction by pure culture was observed for the first time • Strain Ni1-3 effectively reduced bromate under both aerobic and anaerobic conditions • ROS and SOS response genes were strongly induced in the presence of bromate.

[Pyrolysis and gas emissions characteristics of six tree species in Heilongjiang Province, China]. / 黑龙江省6种乔木叶片热解特性及气体释放特征.

Forest fuels are the basis of fire occurrences, while ground dead fuels are an important part of forest fuels. Undestanding the pyrolysis characteristics and gas emissions of forest fuels is of great significance to explore the effects of forest fire on atmospheric environment and carbon balance, as well as to prevent and combat forest fire. In this study, the thermogravimetric analysis and gas emission analysis were conducted on leaf litter of six tree species (Pinus sylvestris var. mongolica, Picea koraiensis, Fraxinus mandshurica, Juglans mandshurica, Quercus mongolica, Betula platyphylla) in Heilongjiang Province to explore the pyrolysis process and combustibility of forest fuels, to analyze their pyrolysis characteristics, pyrolysis kinetics characteristics, gas emission characteristics. A four-dimensional evaluation of their combustibility was conducted based on pyrolysis parameters. The results showed that the pyrolysis temperature of holocellulose in the leaves of those six tree species ranged in 143.31-180.48 ℃ at the beginning and 345.04-394.38 ℃ at the end, lignin pyrolysis temperature ranged in 345.04-394.38 ℃ at the beginning and 582.85-609.31 ℃ at the end. The pyrolysis of the six kinds of arbor blades during the pyrolysis process affected fuel ash content, quality and temperature of the total pyrolysis. The activation energies of two main pyrolysis stages of leaves of six tree species were 18.88-27.08 kJ·mol-1 and 13.25-27.54 kJ·mol-1, respectively, and the pre-exponential factors were 3.13-26.28 min-1 and 1.30-22.55 min-1. The holocellulose activation energy and pre-exponential factor of the pyrolysis stage for P. koraiensis, F. mandshurica, Q. mongolica, and B. platyphylla were greater than that of the lignin pyrolysis stage, while the opposite was true for P. sylvestris var. mongolica and J. mandshurica. The release amounts of CO and CO2 at the pyrolysis stage of the holocellulose was 535.16-880.11 mg·m-3 and 7004.97-10302.05 mg·m-3, and that at the pyrolysis stage of lignin was 240.31-1104.67 mg·m-3 and 20425.60-33946.68 mg·m-3, respectively. The release of CO and CO2 at the pyrolysis stage of healdellulose was less, but mass loss was greater than that at the pyrolysis stage of lignin. In the four-dimensional combustibility ranking of the six tree species leaves, B. platyphylla was the best ignitable, P. koraiensis was the most combustible, and P. sylvestris var. mongolica was the most sustainable and consumable. The ignitability was significantly positively correlated with pyrolysis kinetics parameters of the holocellulose, while the sustainability was negatively correlated with that of lignin.

Comparative study of the physiological responses of Skeletonema costatum and Thalassiosira weissflogii to initial pCO2 in batch cultures, with special reference to bloom dynamics.

Extensive studies have documented the responses of diatoms to environmental drivers in the context of climate change. However, bloom dynamics are usually ignored in most studies. Here, we investigated the effects of the initial pCO2 on the bloom characteristics of two cosmopolitan diatoms, Skeletonema costatum and Thalassiosira weissflogii. Batch cultures with two initial pCO2 conditions (LC: 400 µatm; HC: 1000 µatm) were used to investigate bloom dynamics under current and ocean acidification scenarios. The simulated S. costatum bloom was characterized by fast accumulation, a rapid decline in biomass, and a shorter stationary phase. The T. weissflogii bloom had a longer stationary phase, and cell density remained at high levels after culturing for 19 days. The physiological performances of the two diatoms varied significantly in the different bloom phases. We found that the initial pCO2 has modulating effects on biomass accumulation and bloom dynamics for these two diatoms. The higher initial pCO2 enhanced the specific growth rate of T. weissflogii by 6% in the exponential phase, leading to higher cell densities, while 86% higher decay rates were observed in the HC cultures of S. costatum. Overall, ocean acidification may alter the dynamics of diatom blooms and may have profound impacts on the biological carbon pump.

National Proficiency Testing of Molecular Diagnostics for Tuberculosis and Drug Resistance Detection - China, 2014-2019.

What is already known about this topic? Proficiency testing (PT) is a key component of quality assurance and is essential in ensuring accurate laboratory diagnosis of tuberculosis (TB) and drug-resistant TB. The National Tuberculosis Reference Laboratory (NTRL) developed a novel PT panel to test laboratories' proficiency for molecular TB diagnostic assays throughout the TB laboratory network. A total of 6 PT rounds for molecular diagnostics were conducted by NTRL from 2014 to 2019. What is added by this report? PT conducted using artificial sputum specimens increased from 120 in the first round to 1,835 in the sixth round. Overall, laboratories demonstrated good proficiency for MTB and drug-resistance detection by molecular diagnostics, which is evident from the qualification rates over the six rounds: 95%, 97%, 96%, 93%, 93%, and 97%, respectively. What are the implications for public health practice? The use of artificial sputum specimens for PT panel production to test TB molecular diagnostics in China is feasible. Most of the participating laboratories provided reliable molecular diagnostic results for MTB and drug-resistance detection. The TB laboratory network can be instrumental in implementing PT expansion and improving the quality of TB molecular diagnosis in China.

Syntheses of 4-allyl-/4-allenyl-4-(arylthio)-1,4-dihydroisoquinolin-3-ones via the photochemical Doyle-Kirmse reaction.

Facile synthesis of 4-allyl-/4-allenyl-4-(arylthio)-1,4-dihydroisoquinolin-3-ones via the visible-light-induced Doyle-Kirmse reaction of 4-diazo-1,4-dihydroisoquinolin-3-ones with allyl-/propargyl sulfides is reported. The reaction proceeds via the generation of free carbenes from cyclic diazo compounds followed by in situ formation of sulfonium ylide intermediates, which subsequently undergo [2,3-sigmatropic rearrangement] to give highly functionalized dihydroisoquinolinones in moderate to good yields. Broad substrate scope, and catalyst-free and mild conditions are the merits of this reaction.

Prevalence of Mycobacterium tuberculosis resistant to bedaquiline and delamanid in China.

OBJECTIVES: The new antituberculous drugs delamanid and bedaquiline form the last line of defence against drug-resistant tuberculosis (TB). Understanding the background prevalence of resistance to new drugs can help predict the lifetime of these drugs' effectiveness and inform regimen design. METHODS: Mycobacterium tuberculosis without prior exposure to novel anti-TB drugs were analysed retrospectively. Drug susceptibility testing for bedaquiline, delamanid, linezolid, clofazimine and widely used first- and second-line anti-TB drugs was performed. All TB isolates with resistance to new or repurposed drugs were subjected to whole-genome sequencing to explore the molecular characteristics of resistance and to perform phylogenetic analysis. RESULTS: Overall, resistance to delamanid, bedaquiline, linezolid and clofazimine was observed in 0.7% (11/1603), 0.4% (6/1603), 0.4% (7/1603) and 0.4% (6/1603) of TB isolates, respectively. Moreover, 1.0% (1/102), 2.9% (3/102), 3.9% (4/102) and 1.0% (1/102) of multidrug-resistant TB (MDR-TB) were resistant to bedaquiline, delamanid, linezolid and clofazimine, respectively. Whereas 22.2% (2/9) of extensively-drug resistant tuberculosis (XDR-TB) isolates were resistant to both delamanid and linezolid, and none was resistant to bedaquiline or clofazimine. Phylogenetic analysis showed that recent transmission occurred in two XDR-TB with additional resistance to delamanid and linezolid. None known gene mutation associated with delamanid resistance was detected. All four isolates with cross-resistance to bedaquiline and clofazimine had a detected gene mutation in Rv0678. Three of five strains with linezolid resistance had a detected gene mutation in rplC. CONCLUSION: Detection of resistance to new anti-TB drugs emphasises the pressing need for intensive surveillance for such resistance before their wide usage.

A muscle-epidermis-glia signaling axis sustains synaptic specificity during allometric growth in Caenorhabditis elegans.

Synaptic positions underlie precise circuit connectivity. Synaptic positions can be established during embryogenesis and sustained during growth. The mechanisms that sustain synaptic specificity during allometric growth are largely unknown. We performed forward genetic screens in C. elegans for regulators of this process and identified mig-17, a conserved ADAMTS metalloprotease. Proteomic mass spectrometry, cell biological and genetic studies demonstrate that MIG-17 is secreted from cells like muscles to regulate basement membrane proteins. In the nematode brain, the basement membrane does not directly contact synapses. Instead, muscle-derived basement membrane coats one side of the glia, while glia contact synapses on their other side. MIG-17 modifies the muscle-derived basement membrane to modulate epidermal-glial crosstalk and sustain glia location and morphology during growth. Glia position in turn sustains the synaptic pattern established during embryogenesis. Our findings uncover a muscle-epidermis-glia signaling axis that sustains synaptic specificity during the organism's allometric growth.

Development of a loop-mediated isothermal amplification coupled lateral flow dipstick targeting erm(41) for detection of Mycobacterium abscessus and Mycobacterium massiliense.

Mycobacterium abscessus (M. abscessus) and Mycobacterium massiliense (M. massiliense) are major pathogens that cause post-surgical wound infection and chronic pulmonary disease. Although they are closely related subspecies of M. abscessus complex, their infections are associated with different drug-resistance and cure rate. In the present study, a loop-mediated isothermal amplification (LAMP) coupled with lateral flow dipstick (LFD) method was developed to simultaneous detect M. abscessus and M. massiliense, via specific erm(41) gene. The amplification was carried out at 65 °C for only 60 min, and the results could be visualized on a lateral flow strip. Positive results only occurred in M. abscessus and M. massiliense, no cross-reaction with other mycobacterial species was observed. Therefore, the cost-effective MABC (M. abscessus complex)-LAMP-LFD method developed here was able to correct the diagnose of M. abscessus and M. massiliense infection in a short time. Thus, this method could be used to guide clinicians in treatment of M. abscessus group infections.