Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

Discussion on the Main Control Effect of Geological Structures on Coal and Gas Outburst.

Coal and gas outbursts are great natural disasters in underground coal mines, seriously threatening the lives of miners and coal mine safety. The study of the relationship between coal and gas outbursts and geological structures has always been an important component of gas geological work, and it also serves as a foundation for coal mines to develop gas outburst prevention measures. This study considers groups F and E coal seams, mainly mined in the southwest wing of Likou syncline in the Pingdingshan mining area, as the research object. Based on actual outburst data, this study investigates the distribution and intensity characteristics of coal and gas outbursts and the correlation between coal and gas outbursts and geological structures using gas geological analysis methods and taking structural control as the main line. The results depict that the coal and gas outbursts in the study area have obvious district and subband characteristics. The outburst district mainly occurred in the East, and the four outburst zones, I, II, III, and IV, were the concentrated occurrence subband of coal and gas outbursts. The district and subband of coal and gas outburst are mainly controlled by and related to the geological structure. The eastern fold structure has developed more than the middle and western districts, which is why the eastern part of the coal and gas outburst is more severe than in the middle and western regions. Under the structural background of the coal and gas outburst district, the subband outburst characteristics were mainly controlled by local geological structural conditions.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.

BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Desorption Effects and Laws of Multiscale Gas-Bearing Coal with Different Degrees of Metamorphism.

Understanding gas desorption effects and laws of coal mass under different conditions is essential for the effective exploration of gas emission in underground coal mines, prediction and prevention of coal and gas outburst, accurate detection of gas [coal methane (CBM)] content in coal seams, and prediction of CBM productivity. Using a self-developed test platform, we simulated gas adsorption and desorption and performed physical simulation tests. Based on these tests, we investigated the differences in the total amount of gas desorbed, desorption rate, and initial amount of gas desorbed by long-flame coal, coking coal, meager-lean coal, and anthracite on different scales under different gas pressures. Two methods are used for compensating gas loss, namely, the method and the power function method, as stipulated in the current Standards for Determination of Gas Content in Coal Seams in China. By combining these two methods, we analyzed the applicability of these two compensation methods in coal on different scales with varying degrees of metamorphism under gas pressures. The results demonstrated that (1) under the same gas adsorption pressure, the cumulative total amount of gas desorbed per unit mass within 90 min for the four kinds of coal samples increases with the degree of metamorphism. Changes in the cumulative amount of gas desorbed per unit mass and the desorption rate with the degree of metamorphism vary with stages. Notably, a higher adsorption pressure leads to a more obvious stage change. (2) Under the same gas adsorption pressure, the cumulative total amount of gas desorbed per unit mass and the desorption rate of coal with the same degree of metamorphism are inversely proportional to the size of the coal sample. This indicates significant scale effects. The larger the degree of metamorphism and gas adsorption pressure, the more significant are the scale effects of gas desorption. (3) For coal with the same degree of metamorphism, the higher gas adsorption pressure leads to a larger cumulative total amount of gas desorbed and a higher desorption rate throughout the desorption process and a larger proportion of the cumulative amount of gas desorbed in the initial stage. The smaller the size of the coal sample, the more obvious the pressure effects of gas desorption are. (4) For coal samples with the same degree of metamorphism, when the gas content in coal seams is kept constant, the larger the size of the coal sample, the smaller the actual gas loss is. Moreover, a higher gas content in coal seams results in a greater gas loss and a larger calculation error for gas loss. Compared with the method, the power function method reveals a smaller deviation between the calculated gas loss and the actual gas loss, which is found to be more accurate. A larger size coal sample results in higher accuracy in the calculated gas loss.

Recent Advances of Chitosan-Based Injectable Hydrogels for Bone and Dental Tissue Regeneration.

Traditional strategies of bone repair include autografts, allografts and surgical reconstructions, but they may bring about potential hazard of donor site morbidity, rejection, risk of disease transmission and repetitive surgery. Bone tissue engineering (BTE) is a multidisciplinary field that offers promising substitutes in biopharmaceutical applications, and chitosan (CS)-based bone reconstructions can be a potential candidate in regenerative tissue fields owing to its low immunogenicity, biodegradability, bioresorbable features, low-cost and economic nature. Formulations of CS-based injectable hydrogels with thermo/pH-response are advantageous in terms of their high-water imbibing capability, minimal invasiveness, porous networks, and ability to mold perfectly into an irregular defect. Additionally, CS combined with other naturally-derived or synthetic polymers and bioactive agents has proven to be an effective alternative to autologous bone and dental grafts. In this review, we will highlight the current progress in the development of preparation methods, physicochemical properties and applications of CS-based injectable hydrogels and their perspectives in bone and dental regeneration. We believe this review is intended as starting point and inspiration for future research effort to develop the next generation of tissue-engineering scaffold materials.

[Allelic loss of 6q16.3 microsatellite DNA in childhood acute lymphoblastic leukemia and bioinformatics analysis].

OBJECTIVE: To locate the cluster region of loss of heterozygosity (LOH) in children with acute lymphoblastic leukemia (ALL), and explore the new tumor suppressor gene. METHODS: Allelic loss was analyzed by PCR with 15 microsatellite markers mapping on 6q16.3. The LOH was analyzed by bioinformatics. The relationship between LOH and clinical factors was further analyzed. RESULTS: The frequency of LOH at least at one loci on 6q16.3 was 32.7%. The LOH in relapsed patients was higher than those in not relapsed. The higher frequency of LOH was observed in two regions of D6S1709-D6S1028 and D6S2160-D6S1580 at 6q16.3. GRIK2 may be a candidate of tumor suppressor gene. There are 12 ESTs may carry out new anti-oncogene. Patients with 6q LOH had higher WBC counts (P < 0.01), blast cells percentage (P < 0.01), relapse rate (P < 0.05) and chromosomal aberration (P < 0.05). CONCLUSION: D6S1709-D6S1028 and D6S2160-D6S1580 are two regions of minimus deletion on 6q16.3 in which tumor suppressor gene may exist. The LOH on 6q16.3 may be a prognostic index of children with ALL.