Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor.

The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG3-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG3-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH3 in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG3-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C, and reaction time of 12 h, consequently, achieving a PAL-PEG3-Ph-rhG-CSF product yield of 70.8%. The reaction mixture was purified via preparative liquid chromatography, yielding the single-modified product PAL-PEG3-Ph-rhG-CSF with a HPLC purity exceeding 95%. The molecular weight of PAL-PEG3-Ph-rhG-CSF was 19297 Da by MALDI-TOF-MS, which was consistent with the theoretical value. The circular dichroism analysis revealed no significant change in its secondary structure compared to unmodified rhG-CSF. The PAL-PEG3-Ph-rhG-CSF retained 82.0% of the in vitro biological activity of unmodified rhG-CSF. The pharmacokinetic analyses showed that the serum half-life of PAL-PEG3-Ph-rhG-CSF was 7.404 ± 0.777 h in mice, 4.08 times longer than unmodified rhG-CSF. Additionally, a single subcutaneous dose of PAL-PEG3-Ph-rhG-CSF presented comparable in vivo efficacy to multiple doses of rhG-CSF. This study demonstrated an efficacious strategy for developing long-acting rhG-CSF drug candidates.

The importance of the thickness of femoral lateral wall for treating intertrochanteric fractures: a finite elements analysis.

To explore how the thickness of the femoral lateral wall influences the effectiveness of internal fixation systems used to treat intertrochanteric fractures. CT images of the pelvis and femur of a male adult were used to construct an intertrochanteric fracture model (AO/OTA 31-A2) with various thicknesses of the femoral lateral wall (FLW). Four finite element (FE) models were created with the lateral femoral walls being 10 mm, 20 mm, 30 mm, and 40 mm thick. The fracture models were fixed with a dynamic hip screw (DHS), a proximal femoral nail anti-rotation (PFNA), and a proximal femoral locking compression plate (P-FLCP). A simulated vertical load was applied to the femoral head. The stress and displacement of the implant and femur in each model were recorded for comparison. The FE analysis of the intertrochanteric fracture models showed that the PFNA system could provide better stability than the DHS and P-FLCP with the same thickness of FLW. The FLW provided buttress support to the femoral head and neck when using a DHS and PFNA, and the buttress strength was proportional to the thickness of FLW. The maximum stress in the DHS model was recorded on the DHS plate which accommodated the lag screw. For the PFNA model, the maximum stress appeared at the connection between the nail and blade. In the P-FLCP model, the maximum stresses were highly concentrated at the connection between the cephalic nails and the proximal plate. The thickness of the femoral lateral wall should be considered an important factor when selecting a suitable internal fixation system for intertrochanteric fractures. Based on the FE analysis, intramedullary fixation, such as PFNA, experiences lower stress levels and a moderate displacement in comparison to DHS and P-FCLP when used to treat intertrochanteric fractures.

Transcriptomic analysis of the fat body of resistant and susceptible silkworm strains, Bombyx mori (Lepidoptera), after oral treatment with fenpropathrin.

The widespread use of pyrethroid pesticides has brought serious economic losses in sericulture, but there is still no viable solution. The key to solving the problem is to improve silkworm resistance to pesticides, which depends on understanding the resistance mechanism of silkworms to pesticides. This study aimed to use transcriptomes to understand the underlying mechanism of silkworm resistance to fenpropathrin, which will provide a theoretical molecular reference for breeding pesticide-resistant silkworm varieties. In this study, the fat bodies of two strains with differential resistance after 12 h of fenpropathrin feeding were analyzed using RNA-Seq. After feeding fenpropathrin, 760 differentially expressed genes (DEGs) were obtained in the p50(r) strain and 671 DEGs in the 8y strain. The DEGs involved in resistance to fenpropathrin were further identified by comparing the two strains, including 207 upregulated DEGs in p50(r) and 175 downregulated DEGs in 8y. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these fenpropathrin-related DEGs are mainly enriched in the metabolism and transporter pathways. Moreover, 28 DEGs involved in the metabolic pathway and 18 in the transporter pathway were identified. Furthermore, organic cation transporter protein 6 (BmOCT6), a transporter pathway member, was crucial in enhancing the tolerance of BmN cells to fenpropathrin. Finally, the knockdown of the expression of the homologs of BmOCT6 in Glyphodes pyloalis (G. pyloalis) significantly decreased the resistant level of larvae to fenpropathrin. The findings showed that the metabolism and transporter pathways are associated with resistance to fenpropathrin in silkworm, and OCT6 is an effective and potential target not only for silkworm breeding but also for pest biocontrol.

Uric acid metabolism promotes apoptosis against Bombyx mori nucleopolyhedrovirus in silkworm, Bombyx mori.

The white epidermis of silkworms is due to the accumulation of uric acid crystals. Abnormal silkworm uric acid metabolism decreases uric acid production, leading to a transparent or translucent phenotype. The oily silkworm op50 is a mutant strain with a highly transparent epidermis derived from the p50 strain. It shows more susceptibility to Bombyx mori nucleopolyhedrovirus (BmNPV) infection than the wild type; however, the underlying mechanism is unknown. This study analysed the changes in 34 metabolites in p50 and op50 at different times following BmNPV infection based on comparative metabolomics. The differential metabolites were mainly clustered in six metabolic pathways. Of these, the uric acid pathway was identified as critical for resistance in silkworms, as feeding with inosine significantly enhanced larval resistance compared to other metabolites and modulated other metabolic pathways. Additionally, the increased level of resistance to BmNPV in inosine-fed silkworms was associated with the regulation of apoptosis, which is mediated by the reactive oxygen species produced during uric acid synthesis. Furthermore, feeding the industrial strain Jingsong (JS) with inosine significantly increased the level of larval resistance to BmNPV, indicating its potential application in controlling the virus in sericulture. These results lay the foundation for clarifying the resistance mechanism of silkworms to BmNPV and provide new strategies and methods for the biological control of pests.

[Biomechanical analysis of four internal fixations for Pauwels â ¢ femoral neck fractures with defects].

OBJECTIVE: To investigate the biomechanical characteristics of different internal fixations for Pauwels type Ⅲ femoral neck fracture with defect, and provide reference for the treatment of femoral neck fracture. METHODS: Three-dimensional (3D) finite element models of femoral neck fractures were established based on CT images, including fracture and fracture with defects. Four internal fixations were simulated, namely, inverted cannulated screw(ICS), ICS combined with medial buttress plate, the femoral neck system (FNS) and FNS combined with medial buttress plate. The von Mises stress, model stiffness and fracture displacements of fracture models under 2 100 N axial loads were measured and compared. RESULTS: When femoral neck fracture was fixed by ICS and FNS, the peak stress was mainly concentrated on the surface of the screw near the fracture line, and the peak stress of FNS is higher than that of ICS;When the medial buttress plate was combined, the peak stress was increased and transferred to medial buttress plate, with more obvious of ICS fixation. For the same fracture model, the stiffness of FNS was higher than that of ICS. Compared with femoral neck fracture with defects, fracture model showed higher stiffness in the same internal fixation. The use of medial buttress plate increased model stiffness, but ICS increased more than FNS. The fracture displacement of ICS model exceeded that of FNS. CONCLUSION: For Pauwels type Ⅲ femoral neck fracture with defects, FNS had better biomechanical properties than ICS. ICS combined with medial buttress plate can better enhance fixation stability and non-locking plate is recommended. FNS had the capability of shear resistance and needn't combine with medial buttress plate.

Fumarate mitigates disruption induced by fenpropathrin in the silkworm Bombyx mori (Lepidoptera): A metabolomics study.

The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF-FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro. Therefore, IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.

Bombyx mori transcription factor, E74A, beneficially affects BmNPV infection through direct interaction.

BACKGROUND: Nucleopolyhedrovirus (NPV), one of the baculoviruses, is a promising biopesticide for pest control. Lepidopteran account for 70% of pests, therefore investigation on highly conserved genes associated with viral infections in the lepidopteran model, the silkworm, will serve as a valuable reference for improving the effectiveness of pest management. BmE74A is a member of the erythroblast transformation-specific (ETS) family of transcription factors in Bombyx mori, which we previously found to be highly conserved and closely associated with BmNPV. This study aimed to elucidate the role of BmE74A in viral infection. RESULTS: A significantly high expression of BmE74A in eggs indicated its important role in embryonic development, as did relatively high expressions in the hemolymph and midgut. Significant differences in BmE74A expression in different resistant strains after BmNPV infection suggested its involvement as a response to viral infection. Moreover, RNA interference (RNAi) and overexpression experiments confirmed the important role of BmE74A in promoting viral infection. BmNPV infection was significantly suppressed and enhanced by BmE74A knockdown and overexpression, respectively. Besides, BmE74A was found to regulate the expression of BmMdm2 and Bmp53. Furthermore, the binding of ETS, the functional domain of BmE74A, to occlusion-derived virus proteins was confirmed by far-western blotting, and four viral proteins that may interact with ETS proteins were identified by mass spectrometry. Similarly, a homolog of BmE74A in Spodoptera litura was also found to be involved in larval susceptibility to BmNPV. CONCLUSION: BmE74A promotes BmNPV proliferation by directly interacting with the virus, which may be related to the suppression of the p53 pathway. © 2022 Society of Chemical Industry.

Identification of the in vitro antiviral effect of BmNedd2-like caspase in response to Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection.

The validation of the role of several genes related to Bombyx mori nucleopolyhedrovirus infection in vivo.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of primary silkworm pathogens and causes a serious damage of cocoon losses every year. Recent years, many works have been done to clarify the silkworm anti-BmNPV mechanism, and a significant progress has been made in screening and studying of genes and proteins related to BmNPV infection, but several of them lacked the proofs in vivo. In this study, to further validate the function of seven newly reported genes in vivo, including BmAtlatin-n, Bmferritin-heavy chain (BmFerHCH), Bmthymosin (BmTHY), Bmseroin1, Bmseroin2, Bmnuclear hormone receptors 96 (BmNHR96), and BmE3 ubiquitin-protein ligase SINA-like 10 (BmSINAL10), the response of them in the midgut, fat body, and hemolymph of differentially resistant strains (resistant strain YeA and susceptible strain YeB) at 48 h following BmNPV infection were analyzed. The results showed that the relative stable or upregulated expression level of BmAtlatin-n, BmTHY, Bmseroin1, and Bmseroin2 in YeA resistant strain following BmNPV infection further indicated their antiviral role in vivo, compared with susceptible YeB strain. Moreover, the significant downregulation of BmFerHCH, BmNHR96, and BmSINAL10 in both strains following BmNPV infection revealed their role in benefiting virus infection, as well as the upregulation of BmFerHCH in YeB midgut and BmSINAL10 in YeB hemolymph. These data could be used to complementary the proofs of the function of these genes in response to BmNPV infection.

Geographical isolation and environmental heterogeneity contribute to the spatial genetic patterns of Quercus kerrii (Fagaceae).

Southwest China is one of the major global biodiversity hotspots. The Tanaka line, extending within southwestern China from its northwest to its southeast, is an important biogeographical boundary between the Sino-Japanese and Sino-Himalayan floristic regions. Understanding the evolutionary history of the regional keystone species would assist with both reconstructing historical vegetation dynamics and ongoing biodiversity management. In this research, we combined phylogeographic methodologies and species distribution models (SDMs) to investigate the spatial genetic patterns and distribution dynamics of Quercus kerrii, a dominant evergreen oak inhabiting southwest China lowland evergreen-broadleaved forests (EBLFs). A total of 403 individuals were sampled from 44 populations throughout southwest China. SDMs and mismatch distribution analysis indicated that Q. kerrii has undergone northward expansion since the Last Glacial Maximum (LGM). Quantitative analysis revealed that the range expansion of Q. kerrii since the LGM exceeded that of the sympatric mid-elevation species Quercus schottkyana, likely owing to their contrasting distribution elevations and habitat availabilities. The historical climate change since the LGM and the latitude gradient of the region played an important role in shaping the genetic diversity of Q. kerrii. The genetic differentiation index and genetic distance surface of Q. kerrii populations east of the Tanaka line exceeded those to its west. The long-term geographic isolation and environmental heterogeneity between the two sides of the Tanaka line might increase species divergence patterns and local adaptation. This study provides new insights into the historical dynamics of subtropical EBLFs and the changing biota of southwest China.

Evolutionary response to the Qinghai-Tibetan Plateau uplift: phylogeny and biogeography of Ammopiptanthus and tribe Thermopsideae (Fabaceae).

Previous works resolved diverse phylogenetic positions for genera of the Fabaceae tribe Thermopsideae, without a thoroughly biogeography study. Based on sequence data from nuclear ITS and four cpDNA regions (matK, rbcL, trnH-psbA, trnL-trnF) mainly sourced from GenBank, the phylogeny of tribe Thermopsideae was inferred. Our analyses support the genera of Thermopsideae, with the exclusion of Pickeringia, being merged into a monophyletic Sophoreae. Genera of Sophoreae were assigned into the Thermopsoid clade and Sophoroid clade. Monophyly of Anagyris, Baptisia and Piptanthus were supported in the Thermopsoid clade. However, the genera Thermopsis and Sophora were resolved to be polyphyly, which require comprehensive taxonomic revisions. Interestingly, Ammopiptanthus, consisting of A. mongolicus and A. nanus, nested within the Sophoroid clade, with Salweenia as its sister. Ammopiptanthus and Salweenia have a disjunct distribution in the deserts of northwestern China and the Hengduan Mountains, respectively. Divergence age was estimated based on the ITS phylogenetic analysis. Emergence of the common ancestor of Ammopiptanthus and Salweenia, divergence between these two genera and the split of Ammopiptanthus species occurred at approximately 26.96 Ma, 4.74 Ma and 2.04 Ma, respectively, which may be in response to the second, third and fourth main uplifts of the Qinghai-Tibetan Plateau, respectively.

Evolutionary History of a Desert Shrub Ephedra przewalskii (Ephedraceae): Allopatric Divergence and Range Shifts in Northwestern China.

Based on two chloroplast DNA sequences, psbA-trnH and trnT-trnF, phylogeographical patterns of a desert shrub, Ephedra przewalskii, were examined across most of its geographic range in northwestern China. A total of sixteen haplotypes were detected. There was a common haplotype in each basin, that was haplotype A in Tarim Basin, haplotype G in Junggar Basin, and haplotype M in Qaidam Basin. Genetic variance mainly occurred among populations, geographic regions, and eleven geographic groups subdivided by SAMOVA analysis. E. przewalskii likely had a smaller and more fragmented geographic range during the Last Glacial Maximum, which was determined based on ecological niche modelling. Three groups of E. przewalskii populations were detected to have experience range expansion, and this was based on significant values of Fu's FS, Tajima's D, and unimodel mismatch distributions. The cold and dry climate during the glacial period of the Quaternary is postulated to have been a driver for significant genetic isolation and divergence among populations or groups in E. przewalskii, whereas the warmer and wetter climate during the interglacial period is speculated to have provided favourable conditions for range expansion of the species.