Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.

Manipulation of Triplet Excited States in Two-Component Systems for High-Performance Organic Afterglow Materials.

The past several years have witnessed the tremendous development of novel chemical structures, new design strategies and intriguing applications in the field of room-temperature phosphorescence (RTP) and organic afterglow materials. This Review article focuses on recent advancements of high-performance organic afterglow materials obtained by two-component design strategies such as a dopant-matrix, donor-acceptor, sensitization, and energy-transfer strategies. Based on some cutting-edge studies, organic afterglow efficiency has been largely improved, exceeding 90 % in several cases. Organic afterglow durations reach tens of seconds in phosphorescence systems and hours in donor-acceptor systems. Organic afterglow brightness outcompetes some inorganic afterglow materials in the first several seconds after ceasing excitation source. Organic afterglow colors cover the whole visible regions and extend to near-infrared regions with respectful afterglow efficiency. On the basis of these achievements, researchers demonstrate promising applications of organic afterglow materials in diverse fields, which has also been reviewed.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.

BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome. RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

Manipulation of Organic Afterglow by Thermodynamic and Kinetic Control.

The fabrication of room-temperature organic phosphorescence and afterglow materials, as well as the transformation of their photophysical properties, has emerged as an important topic in the research field of luminescent materials. Here, we report the establishment of energy landscapes in dopant-matrix organic afterglow systems where the aggregation states of luminescent dopants can be controlled by doping concentrations in the matrices and the methods of preparing the materials. Through manipulation by thermodynamic and kinetic control, dopant-matrix afterglow materials with different aggregation states and diverse afterglow properties can be obtained. The conversion from metastable aggregation state to thermodynamic stable aggregation state of the dopant-matrix afterglow materials to leads to the emergence of intriguing afterglow transformation behavior triggered by thermal and solvent annealing. The thermodynamically unfavorable reversible afterglow transformation process can also be achieved by coupling the dopant-matrix afterglow system to mechanical forces.

Identification of a new GLDC gene alternative splicing variant and its protumorigenic roles in lung cancer.

Aim: To clarify the regulatory roles of GLDCV1, the first identified truncated glycine decarboxylase (GLDC), on cancer stem cells and tumorigenesis. Materials & methods: RT-PCR or RT-qPCR, immunoblotting and immunohistochemical staining were applied to assess gene expression. MTT, BrdU incorporation and colony formation assays were used to examine cell proliferation capacity. Soft agar colony formation and in vivo transplantation were applied to evaluate cellular transformation and tumorigenesis. Results & conclusion: Expression of GLDCV1 or GLDC was enhanced in non-small-cell lung cancer cell line and clinical samples. GLDCV1 overexpression induced MRC5 cell proliferation, transformation and tumorigenesis. Additionally, GLDCV1 increased lactate production and cancer stem cell marker expression and activated ERK and P38 pathways. Our study gained deeper insight into GLDC oncogene.

An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.

The regulatory function of Fbxo40 has been well characterized in mice. As a key component of the SCF-E3 ubiquitin ligase complex, Fbxo40 induces IRS1 ubiquitination, thus inactivating the IGF1/Akt pathway. The expression of Fbxo40 is restricted to muscle, and mice with an Fbxo40 null mutation exhibit muscle hypertrophy. However, the function of FBXO40 has not been elucidated in pigs, and it is not known whether FBXO40 mutations affect their health. We therefore generated FBXO40 knockout pigs using somatic cell nuclear transfer (SCNT) technology. CRISPR/Cas9 technology was combined with G418 selection, making it possible to generate donor cells at an efficiency of 75.86%. In muscle from FBXO40 knockout pigs, IRS1 levels were higher, and the IGF1/Akt pathway was stimulated. Mutant animals also had approximately 4% more muscle mass compared to WT controls. The knockout pigs developed normally and no pathological changes were found in major organs. These results demonstrate that FBXO40 is a promising candidate gene for improving production traits in agricultural livestock and for developing therapeutic interventions for muscle diseases.

Comparison of staggered and conventional posterior single-door laminoplasty.

OBJECTIVE: This study aimed to delineate the clinical and radiological outcomes between two different single-door laminoplasty techniques, the staggered approach and the conventional one-sided approach, in treating cervical spondylotic myelopathy (CSM). METHODS: This is a retrospective chart review that involved 67 patients who had CSM with symptoms lasting for ≥3 months, and underwent staggered laminoplasty (Group A, n=35) or conventional laminoplasty (Group B, n=32). Outcomes measures included intraoperative parameters, the Japanese Orthopaedic Association (JOA) score, visual analog scale (VAS) for pain, cervical curvature, cervical range of motion (ROM), and radiographic parameters that reflected the level of post-operative muscle atrophy. Follow-up assessments were available at 3-, 6-, and 12-months post-operation. RESULTS: The mean ages in Group A and Group B were 57.11 (SD, 8.02) and 55.28 (SD, 8.47) years, respectively, with a gender distribution of 40.00% female in Group A and 40.63% in Group B (P>0.05). The average operative times were 130.86 (SD, 11.80) and 129.84 (SD, 10.51) minutes, respectively (P>0.05). However, intraoperative blood loss in milliliters was significantly higher in Group A (196.06; SD, 32.69) compared to Group B (155.03; SD, 37.80) (P<0.001). JOA scores revealed no significant post-operative differences between the two groups. Nevertheless, Group A exhibited less VAS pain, reduced post-operative ROM loss at 6 and 12 months, and less alteration in cervical curvature and decreased severity in muscle atrophy at 3-, 6-, and 12-months post-surgery. CONCLUSION: Patients who underwent staggered single-door laminoplasty experienced more favorable outcomes in some metrics than those who received the conventional technique.

Disruption of Zfh3 abolishes mulberry-specific monophagy in silkworm larvae.

Feeding behavior is critical for insect survival and fitness. Most researchers have explored the molecular basis of feeding behaviors by identifying and elucidating the function of olfactory receptors (ORs) and gustatory receptors (GRs). Other types of genes, such as transcription factors, have rarely been investigated, and little is known about their potential roles. The silkworm (Bombyx mori) is a well-studied monophagic insect which primarily feeds on mulberry leaves, but the genetic basis of its monophagy is still not understood. In this report, we focused on a transcription factor encoded by the Zfh3 gene, which is highly expressed in the silkworm central and peripheral nervous systems, including brain, antenna, and maxilla. To investigate its function, Zfh3 was abrogated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) mutagenesis. Since Zfh3 knockout homozygotes are not viable, we studied feeding behavior in heterozygotes, and found that disruption of Zfh3 affects both gustation and olfaction. Mutant larvae lose preference for mulberry leaves, acquire the ability to consume an expanded range of diets, and exhibit improved adaptation to the M0 artificial diet, which contains no mulberry leaves. These results provide the first demonstration that a transcription factor modulates feeding behaviors in an insect.

A dynamic model of coalbed methane emission from boreholes in front of excavation working face: numerical model and its application.

China's current energy consumption is primarily fueled by coal, increasing coal mining with growing energy demand. Coal and gas outburst accidents are common problems in coal mining, and prediction methods are fundamental for preventing such accidents. The gas emission characteristics of boreholes are a combination of comprehensive coal properties and coal seam gas occurrence status; thus, the accurate prediction of gas emissions from boreholes is crucial for preventing such hazards. This paper presents a method for measuring the gas flow rate in continuous boreholes, which is used to predict outburst danger in front of the working face. The model was compared with field measurement data and found suitable for research. The effects of different initial gas pressures, different borehole radius, and different burial depths on gas emissions from boreholes were studied. The results showed that (1) initial gas pressure is the main influencing factor of gas gushing. The amount of gas emission during drilling and the attenuation of gas pressure are more sensitive to pressure. An increase in gas pressure considerably increases the amount of gas gushing out of drilling holes. (2) The increase in the drilling radius increases the generation of coal cuttings, the area of the drilling hole wall, and the degree of damage to the drilling hole wall. Consequently, the amount of gas gushing out of the drilling hole increases. (3) In situ stress occurs mainly because of the increase in gas pressure with an increase in burial depth and the increase in gas desorption caused by the increase in damage to the borehole wall. This study provides a new outburst prediction method, which involves identifying outburst hazards through the gas gushing out of the borehole. The results are expected to aid the control of underground coal and gas outbursts and ensure the safe production of coal mines.

Transplantation of neural stem progenitor cells from different sources for severe spinal cord injury repair in rat.

Neural stem progenitor cell (NSPC) transplantation has been regarded as a promising therapeutic method for spinal cord injury (SCI) repair. However, different NSPCs may have different therapeutic effects, and it is therefore important to identify the optimal NSPC type. In our study, we compared the transcriptomes of human fetal brain-derived NSPCs (BNSPCs), spinal cord-derived NSPCs (SCNSPCs) and H9 embryonic stem-cell derived NSPCs (H9-NSPCs) in vitro and subsequently we transplanted each NSPC type on a collagen scaffold into a T8-9 complete SCI rat model in vivo. In vitro data showed that SCNSPCs had more highly expressed genes involved in nerve-related functions than the other two cell types. In vivo, compared with BNSPCs and H9-NSPCs, SCNSPCs exhibited the best therapeutic effects; in fact, SCNSPCs facilitated electrophysiological and hindlimb functional recovery. This study demonstrates that SCNSPCs may be an appropriate candidate cell type for SCI repair, which is of great clinical significance.

Optimized, visible light-induced crosslinkable hybrid gelatin/hyaluronic acid scaffold promotes complete spinal cord injury repair.

Severe microenvironmental changes after spinal cord injury (SCI) present serious challenges in neural regeneration and tissue repair. Gelatin (GL)- and hyaluronic acid (HA)-based hydrogels are attractive scaffolds because they are major components of the extracellular matrix and can provide a favorable adjustable microenvironment for neurogenesis and motor function recovery. In this study, three-dimensional hybrid GL/HA hydrogel scaffolds were prepared and optimized. The hybrid hydrogels could undergoin situgelation and fit the defects perfectly via visible light-induced crosslinking in the complete SCI rats. We found that the transplantation of the hybrid hydrogel scaffold significantly reduced the inflammatory responses and suppressed glial scar formation in an HA concentration-dependent manner. Moreover, the hybrid hydrogel with GL/HA ratios less than 8/2 effectively promoted endogenous neural stem cell migration and neurogenesis, as well as improved neuron maturation and axonal regeneration. The results showed locomotor function improved 60 days after transplantation, thus suggesting that GL/HA hydrogels can be considered as a promising scaffold for complete SCI repair.

Bmmp influences wing morphology by regulating anterior-posterior and proximal-distal axes development.

Insect wings are subject to strong selective pressure, resulting in the evolution of remarkably diverse wing morphologies that largely determine flight capacity. However, the genetic basis and regulatory mechanisms underlying wing size and shape development are not well understood. The silkworm Bombyx mori micropterous (mp) mutant exhibits shortened wing length and enlarged vein spacings, albeit without changes in total wing area. Thus, the mp mutant comprises a valuable genetic resource for studying wing development. In this study, we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp. Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in noncoding regions. These mutations resulted in decreased Bmmp messenger RNA levels and changes in transcript isoform composition. Bmmp null mutants were generated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 and exhibited changed wing shape, similar to mp mutants, and significantly smaller total wing area. By examining the expression of genes critical to wing development in wildtype and Bmmp null mutants, we found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axes development. We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila. The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity. Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.

Transplantation of collagen sponge-based three-dimensional neural stem cells cultured in a RCCS facilitates locomotor functional recovery in spinal cord injury animals.

Numerous studies have indicated that microgravity induces various changes in the cellular functions of neural stem cells (NSCs), and the use of microgravity to culture tissue engineered seed cells for the treatment of nervous system diseases has drawn increasing attention. The goal of this study was to verify the efficacy of collagen sponge-based 3-dimensional (3D) NSCs cultured in a rotary cell culture system (RCCS) in treating spinal cord injury (SCI). The Basso-Beattie-Bresnahan score, inclined plane test, and electrophysiology results all indicated that 3D cultured NSCs cultured in a RCCS had better therapeutic effects than those cultured in a traditional cell culture environment, suggesting that the microgravity provided by the RCCS could enhance the therapeutic effect of 3D cultured NSCs. Our study indicates the feasibility of combining the RCCS with collagen sponge-based 3D cell culture for producing tissue engineered seed cells for the treatment of SCI. This novel and effective method shows promise for application in cell-based therapy for SCI in the future.

Boosting Organic Afterglow Performance via a Two-Component Design Strategy Extracted from Macromolecular Self-Assembly.

Because intersystem crossing and phosphorescence decay are spin-forbidden in organic systems, it is challenging to obtain high-performance organic afterglow materials. Inspired by two-component design strategy from macromolecular self-assembly, here we report the utilization of synthetic polymers to control the excited state properties of difluoroboron ß-diketonate (BF2bdk) and deuterated BF2bdk compounds for the fabrication of room-temperature organic afterglow materials. The polymer component can interact with BF2bdk excited states by dipole-dipole interactions, lower BF2bdk S1 levels with insignificant effect on T1 levels, reduce ΔEST, and thus enhance intersystem crossing of BF2bdk excited states. The polymer component can also suppress intramolecular motion of BF2bdk triplets and protect BF2bdk triplets from oxygen quenching. The obtained BF2bdk-polymer afterglow materials exhibit emission lifetimes up to 2.2 s and high photoluminescence quantum yields under ambient conditions, display excellent processability and flexibility, and can function as efficient donors for excited state energy transfer to construct red afterglow materials.

Correction: Transplantation of collagen sponge-based three-dimensional neural stem cells cultured in a RCCS facilitates locomotor functional recovery in spinal cord injury animals.

Correction for 'Transplantation of collagen sponge-based three-dimensional neural stem cells cultured in a RCCS facilitates locomotor functional recovery in spinal cord injury animals' by Yunlong Zou et al., Biomater. Sci., 2022, 10, 915-924. DOI: 10.1039/D1BM01744F.

High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation.

The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

A novel worm-like micelles@MOFs precursor for constructing hierarchically porous CoP/N-doped carbon networks towards efficient hydrogen evolution reaction.

Constructing electrocatalysts with plentiful active sites, great mass transfer ability, and high electrical conductivity is critical to realize efficient hydrogen evolution reaction (HER). Hierarchically porous cobalt phosphide/N-doped nanotubular carbon networks (CoP/NCNs) that have all the features were fabricated in this work. For the fabrication, the polymeric worm-like micelles (PWs) with a large aspect ratio were coated by a uniform nanolayer of Zn-Co zeolitic imidazolate frameworks (Zn-Co-ZIFs) on their surface, resulting in the hybrid nanofibers PWs@Zn-Co-ZIFs (HPWs). Inheriting the randomly curved and entanglement properity of PWs, the rigid HPWs formed hybrid networks with the packing voids sized tens to 200 nm. Then, the hybrid networks were treated by pyrolysis-oxidation-phosphidation and ZnO-removal processes, leading to the hierarchically porous CoP/NCNs. In the CoP/NCNs, there are plentiful CoP nanoparticles embedded on the surface of conductive carbon network and fully exposed. When used for HER electrocatalyst, the CoP/NCNs only need small overpotentials (98 and 118 mV in acid and alkaline electrolyte) at 10 mA cm-2. This novel strategy is instructive for tailoring hierarchically porous transition metal phosphide/carbon nanocomposites as promising electrocatalysts.

The Rotary Cell Culture System increases NTRK3 expression and promotes neuronal differentiation and migratory ability of neural stem cells cultured on collagen sponge.

BACKGROUND: Recently, neural stem cell (NSC) therapy has shown promise for the treatment of many neurological diseases. Enhancing the quality of implanted cells and improving therapeutic efficacy are currently research hotspots. It has been reported that collagen sponge material provided sufficient room for cell growth in all directions and promoted the absorption of nutrients and removal of wastes. And also, the Rotary Cell Culture System (RCCS), which mimics the microgravity environment, can be used to culture cells for tissue engineering. MATERIALS AND METHODS: We performed the mRNA and miRNA sequencing to elucidate the regulatory mechanism of NSCs cultured on the collagen sponge in the RCCS system. The luciferase assay and Western blot revealed a direct regulatory role between let-7i-5p and neurotrophic receptor tyrosine kinase 3 (NTRK3; also called TrkC). And then, the neural differentiation markers Tuj1 and Map2 were detected by immunofluorescence staining. In the meantime, the migratory ability of NSCs was detected both in vitro and in spinal cord injury animals. RESULTS: In this study, we demonstrated that the expression of NTRK3 was elevated in NSCs cultured on collagen sponge in the RCCS system. Furthermore, increased NTRK3 expression was regulated by the downregulation of let-7i-5p. Compared to traditionally cultured NSCs, the NSCs cultured on collagen sponge in the RCCS system exhibited better neuronal differentiation and migratory ability, especially in the presence of NT-3. CONCLUSIONS: As the biological properties and quality of transplanted cells are critical for therapeutic success, the RCCS system combined with the collagen sponge culture system shows promise for applications in clinical practice in the future.

Establishment and characterization of immortalized porcine neonatal hepatocytes without the use of viral components.

Porcine hepatocytes are a promising option for xenotransplantation in light of the critical shortage of orthotopic donor livers. Because primary hepatocytes have limited ability to proliferate in vitro, several immortalized hepatocyte lines have been established. However, these cells have typically been generated using a virus-dependent transfection methodology and express viral oncogenes that introduce potential risks in clinical applications. In our study, we established immortalized porcine neonatal hepatocytes by introduction of a plasmid-based hTERT gene expression system by electroporation, without the use of viral components. We detected stable expression of hTERT by RT-PCR and Western blot. The immortalized hepatocytes exhibit a high growth rate, but retain the normal morphology of freshly isolated primary hepatocytes. To date, these immortalized hepatocytes have been expanded for over 80 passages. In addition, no significant differences were detected in glycogen synthesis, secretion of serum albumin, or lipid accumulation between the primary hepatocytes and our immortalized hepatocytes. The cells also exhibit serum-dependent growth and have no capacity for anchorage-independent growth in vitro, demonstrating that they have not been transformed in vitro. Our immortalized porcine hepatocytes will be useful for elucidating the pathogenesis of liver disease and developing efficient treatments. Furthermore, these immortalized hepatocytes may provide a safer source of cells for application in xenotransplantation, compared with immortalized cells generated using viral components.

Comparison of Regenerative Effects of Transplanting Three-Dimensional Longitudinal Scaffold Loaded-Human Mesenchymal Stem Cells and Human Neural Stem Cells on Spinal Cord Completely Transected Rats.

Stem cell-based therapy has been considered as a potential treatment to restore spinal cord injury (SCI) through reconstructing neural networks and providing a favorable microenvironment for neuronal survival, differentiation, and axonal outgrowth. Biomaterial scaffolds can promote cell attachment and survival, neuronal differentiation, and axonal outgrowth; therefore, they were used to combine with stem cells for implantation in SCI treatment. In addition, a longitudinal scaffold can guide regenerated axons with orientated growth and axial extension. Both human umbilical cord-derived mesenchymal stem cells (hMSCs) and human fetal spinal cord-derived neural stem cells (hNSCs) have been applied in clinical trials worldwide. To our knowledge, a parallel comparison of the therapeutic effects of hMSC and hNSC implantations has not been conducted. Hence, in this study, we grafted hMSCs or hNSCs seeded on longitudinal collagen sponge scaffolds into rats with completely transected SCI to examine differences in SCI repair. Both hMSCs and hNSCs had equivalent effects on reducing glial scar formation around the lesion gap. More neuronal class III ß-tubulin-positive neurons and neurofilament-positive nerve fibers were found in the lesion cavity after hNSC implantation. In addition, hNSCs had better capabilities to improve motor function, attenuate inflammation, and promote cell survival than hMSCs. These encouraging results provide a clinical basis for future stem cell-based SCI therapies.