Analysis of influencing factors of HPV vaccination willingness of female sex workers in urban entertainment venues based on the IMB model in Guangxi, China.

OBJECTIVE: Understanding HPV vaccination willingness and its influencing factors among female sex workers (FSWs) in entertainment venues in an urban area of Guangxi, China. METHODS: From 15 August to 15 October 2022, FSWs in entertainment venues with commercial sex trade in an urban area of Guangxi were selected as the study subjects for the questionnaire survey using the method of intentional sampling. The questionnaire based on the information-motivation-behavior (IMB) skills model was used to collect the basic characteristics, HPV and HPV vaccine-related information and cognition, motivation to vaccinate, behavioral skills and willingness to vaccinate from the research targets. A multifactor logistic regression model was used to analyze the factors influencing the research targets' willingness to receive HPV vaccination. RESULTS: Of the 921 research targets, 712 (77.31%) were willing to receive HPV vaccination. The higher the level of knowledge regarding HPV and HPV vaccine-related information, the higher the motivation for HPV vaccination. In addition, the higher the behavioral skills score, the higher the willingness of FSWs in entertainment venues to receive HPV vaccination (P<0.001). FSWs in entertainment venues with lower venue grades [OR(95% CI)=0.693 (0.539, 0.891), P=0.004] were more reluctant to receive HPV vaccination. Those who favored the effectiveness of the vaccine in preventing the disease [OR(95% CI)=2.144 (1.449, 3.174), P<0.001] and those who had heard of HPV vaccine [OR(95% CI)=2.105 (1.451, 3.054), P<0.001], were able to perceive the benefits of HPV vaccination [OR(95% CI)=1.134 (1.045, 1.230), P=0.002]. These individuals acquired greater behavioral skills i.e., self-decision making for HPV vaccination [OR(95% CI)=1.130 (1.008, 1.267), P=0.036] and self-efficacy [OR(95% CI)=1.135 (1.081, 1.191), P<0.001] and they were more willing to receive HPV vaccine. CONCLUSIONS: There was a relatively high HPV vaccination willingness among FSWs in entertainment venues in an urban area of Guangxi, China. Attention should be focused on introducing the benefits of primary prevention measures such as the HPV vaccine for individuals and behavioral skills for HPV vaccination in order to increase their willingness to be vaccinated thus increasing their HPV vaccination rate.

Microsporidia dressing up: the spore polaroplast transport through the polar tube and transformation into the sporoplasm membrane.

Microsporidia are obligate intracellular parasites that infect a wide variety of hosts including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast, and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming a hollow polar tube that transports the sporoplasm components including the nucleus into the host cell. Due to the complicated topological changes occurring in this process, the details of sporoplasm formation are not clear. Our data suggest that the limiting membrane of the nascent sporoplasm is formed by the polaroplast after microsporidian germination. Using electron microscopy and 1,1'-dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine perchlorate staining, we describe that a large number of vesicles, nucleus, and other cytoplasm contents were transported out via the polar tube during spore germination, while the posterior vacuole and plasma membrane finally remained in the empty spore coat. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were also found to localize in the region of the polaroplast and posterior vacuole in mature spores and in the discharged polar tube, which suggested that the polaroplast during transport through the polar tube became the limiting membrane of the sporoplasm. The analysis results of Golgi-tracker green and Golgi marker protein syntaxin 6 were also consistent with the model of the transported polaroplast derived from Golgi transformed into the nascent sporoplasm membrane.IMPORTANCEMicrosporidia, which are obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection by the microsporidia is their unique invasion apparatus which includes the polar filament, polaroplast, and posterior vacuole. When the mature spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that transports the sporoplasm components including the microsporidian nucleus into host cells. Details of the structural difference between the polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting the nucleus and other sporoplasm components. Furthermore, our results indicate that the transported polaroplast transforms into the sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of the polar tube and origin of the sporoplasm membrane, which provide important clarification of the microsporidian infection mechanism.

Effects of isotropic strain on the structure and transport properties of half-Heusler alloy BiBaK: a first-principles investigation.

In this study, using density functional and Boltzmann transport theories, we systematically investigated the effects of tensile and compressive strains on the elastic properties, phonon dispersion relation, electronic structure, and transport properties of the half-Heusler compound BiBaK. We calculated the elastic constants and phonon dispersion curves for BiBaK, which demonstrated its mechanical and thermodynamic stability, respectively, under different isotropic strains. Further, calculations showed that the electronic structure and energy bandgap of BiBaK changed with the application of isotropic strain. A high power factor and low thermal conductivity are key to improving the performance of thermoelectric materials. The figure of merit of BiBaK is 0.6 when it is unstrained and reaches a maximum value of 0.93 at -9% compressive strain and a temperature of 1200 K, indicating that under isotropic compressive strain, BiBaK compounds are efficient thermoelectric materials for high-temperature applications.

Harnessing multiplex crRNA enables an amplification-free/CRISPR-Cas12a-based diagnostic methodology for Nosema bombycis.

IMPORTANCE: The multiplex-crRNA CRISPR/Cas12a detection method saves hands-on time, reduces the risk of aerosol pollution, and can be directly applied to detecting silkworms infected with Nosema bombycis. This study provides a new approach for the inspection and quarantine of silkworm pébrine disease in sericulture and provides a new method for the detection of other pathogens.

Microsporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission.

Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission.

First identification and coinfection detection of Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp. and Giardia duodenalis in diarrheic pigs in Southwest China.

BACKGROUND: Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods. RESULTS: A total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ2 = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ2 = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens. CONCLUSIONS: This study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.

Tight Binding and Dual Encapsulation Enabled Stable Thick Silicon/Carbon Anode with Ultrahigh Volumetric Capacity for Lithium Storage.

Silicon (Si) is regarded as one of the most promising anode materials for high-performance lithium-ion batteries (LIBs). However, how to mitigate its poor intrinsic conductivity and the lithiation/delithiation-induced large volume change and thus structural degradation of Si electrodes without compromising their energy density is critical for the practical application of Si in LIBs. Herein, an integration strategy is proposed for preparing a compact micron-sized Si@G/CNF@NC composite with a tight binding and dual-encapsulated architecture that can endow it with superior electrical conductivity and deformation resistance, contributing to excellent cycling stability and good rate performance in thick electrode. At an ultrahigh mass loading of 10.8 mg cm-2 , the Si@G/CNF@NC electrode also presents a large initial areal capacity of 16.7 mA h cm-2 (volumetric capacity of 2197.7 mA h cm-3 ). When paired with LiNi0.95 Co0.02 Mn0.03 O2 , the pouch-type full battery displays a highly competitive gravimetric (volumetric) energy density of ≈459.1 Wh kg-1 (≈1235.4 Wh L-1 ).

Characterization and Biological Activities of Melanin from the Medicinal Fungi Ophiocordyceps sinensis.

Melanin is a complex natural pigment that is widely present in fungi. The mushroom Ophiocordyceps sinensis has a variety of pharmacological effects. The active substances of O. sinensis have been extensively studied, but few studies have focused on the O. sinensis melanin. In this study, the production of melanin was increased by adding light or oxidative stress, namely, reactive oxygen species (ROS) or reactive nitrogen species (RNS), during liquid fermentation. Subsequently, the structure of the purified melanin was characterized using elemental analysis, ultraviolet-visible absorption spectrum, Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and pyrolysis gas chromatography and mass spectrometry (Py-GCMS). Studies have shown that O. sinensis melanin is composed of C (50.59), H (6.18), O (33.90), N (8.19), and S (1.20), with maximum absorbance at 237 nm and typical melanin structures such as benzene, indole, and pyrrole. Additionally, the various biological activities of O. sinensis melanin have been discovered; it can chelate heavy metals and shows a strong ultraviolet-blocking ability. Moreover, O. sinensis melanin can reduce the levels of intracellular reactive oxygen species and counteract the oxidative damage of H2O2 to cells. These results can help us to develop applications of O. sinensis melanin in radiation resistance, heavy metal pollution remediation, and antioxidant use.

The development of single-chain antibody anchored on the BmE cell membrane to inhibit BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat to sericulture production, and traditional sanitation practices remain the main strategy for controlling BmNPV infection. Although RNAi targeting BmNPV genes engineered into transgenic silkworms has shown to be a promising approach in reducing viral infection, it cannot block viral entry into host cells. Therefore, there is an urgent need to develop new effective prevention and control measures. In this study, we screened a monoclonal antibody 6C5 that potently neutralizes BmNPV infection by clamping the internal fusion loop of the BmNPVglycoprotein64 (GP64). Furthermore, we cloned the VH and VL fragments of mAb-6C5 from the hybridoma cell, and the eukaryotic expression vector of scFv6C5 was constructed to anchor the antibody on the cell membrane. The GP64 fusion loop antibody-expressing cells exhibited a reduced capacity for BmNPV infection. The results from our study provide a novel BmNPV control strategy and lay the foundation for the future development of transgenic silkworms with improved antiviral efficacy.

Stable reference gene selection for Ophiocordyceps sinensis gene expression studies under different developmental stages and light-induced conditions.

The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.

Exploring structural, mechanical, and thermoelectric properties of half-Heusler compounds RhBiX (X = Ti, Zr, Hf): A first-principles investigation.

In this study, the full potential linearization enhanced plane wave method in density functional theory is used. Additionally, the structure, mechanical, and thermoelectric properties of half-Heusler compounds RhBiX (X = Ti, Zr, Hf) are investigated for the first time. The indirect semiconductors RhBiTi and RhBiZr have 0.89 and 1.06 eV bandgap energies, respectively. In contrast, RhBiHf is a direct bandgap semiconductor with a bandgap energy of 0.33 eV. The thermoelectric parameters such as Seebeck coefficient, power factor, electronic conductivity, lattice thermal conductivity, electronic thermal conductivity, and figure of merit ZT, are studied with the semi-classical Boltzmann transport theory. When T = 300 K, RhBiTi, RhBiZr, and RhBiHf show small lattice thermal conductivities, i.e., 10.60, 10.15, and 7.71 W mK-1, respectively, which are consistent with related other studies. The maximum ZT values of RhBiTi, RhBiZr, and RhBiXHf are 0.91, 0.94, and 0.79 at 900 K, respectively. Furthermore, we observed that RhBiX (X = Ti, Zr, Hf) alloy is a thermoelectric material with great potential.

Comparative and Functional Analyses Reveal Conserved and Variable Regulatory Systems That Control Lasalocid Biosynthesis in Different Streptomyces Species.

Lasalocid, a representative polyether ionophore, has been successfully applied in veterinary medicine and animal husbandry and also displays promising potential for cancer therapy. Nevertheless, the regulatory system governing lasalocid biosynthesis remains obscure. Here, we identified two conserved (lodR2 and lodR3) and one variable (lodR1, found only in Streptomyces sp. strain FXJ1.172) putative regulatory genes through a comparison of the lasalocid biosynthetic gene cluster (lod) from Streptomyces sp. FXJ1.172 with those (las and lsd) from Streptomyces lasalocidi. Gene disruption experiments demonstrated that both lodR1 and lodR3 positively regulate lasalocid biosynthesis in Streptomyces sp. FXJ1.172, while lodR2 plays a negative regulatory role. To unravel the regulatory mechanism, transcriptional analysis and electrophoretic mobility shift assays (EMSAs) along with footprinting experiments were performed. The results revealed that LodR1 and LodR2 could bind to the intergenic regions of lodR1-lodAB and lodR2-lodED, respectively, thereby repressing the transcription of the lodAB and lodED operons, respectively. The repression of lodAB-lodC by LodR1 likely boosts lasalocid biosynthesis. Furthermore, LodR2 and LodE constitute a repressor-activator system that senses changes in intracellular lasalocid concentrations and coordinates its biosynthesis. LodR3 could directly activate the transcription of key structural genes. Comparative and parallel functional analyses of the homologous genes in S. lasalocidi ATCC 31180T confirmed the conserved roles of lodR2, lodE, and lodR3 in controlling lasalocid biosynthesis. Intriguingly, the variable gene locus lodR1-lodC from Streptomyces sp. FXJ1.172 seems functionally conserved when introduced into S. lasalocidi ATCC 31180T. Overall, our findings demonstrate that lasalocid biosynthesis is tightly controlled by both conserved and variable regulators, providing valuable guidance for further improving lasalocid production. IMPORTANCE Compared to its elaborated biosynthetic pathway, the regulation of lasalocid biosynthesis remains obscure. Here, we characterize the roles of regulatory genes in lasalocid biosynthetic gene clusters of two distinct Streptomyces species and identify a conserved repressor-activator system, LodR2-LodE, which could sense changes in the concentration of lasalocid and coordinate its biosynthesis with self-resistance. Furthermore, in parallel, we verify that the regulatory system identified in a new Streptomyces isolate is valid in the industrial lasalocid producer and thus applicable for the construction of high-yield strains. These findings deepen our understanding of regulatory mechanisms involved in the production of polyether ionophores and provide novel clues for the rational design of industrial strains for scaled-up production.

Detection of the pathogenic fungus Cordyceps farinosa in the Thitarodes armoricanus soil-rearing environment based on nucleic acid targets.

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation.

Study of Pathogenesis Using Fluorescent Strain of Cordyceps farinosa Revealed Infection of Thitarodes armoricanus Larvae via Digestive Tract.

Cordyceps farinosa is often utilized as a biocontrol agent because of its wide host range, strong lethality, and safety for mammals. Artificial rearing of Thitarodes armoricanus larvae is a prerequisite for the artificial cultivation of Chinese cordyceps, and C. farinosa is the most lethal pathogenic fungus during the rearing process. However, the infection process of C. farinosa is still unclear. In this study, we cloned the promoter of the C. farinosa glyceraldehyde 3-phosphate dehydrogenase gene, constructed the EGFP expression cassette, and integrated it into the C. farinosa genome via Agrobacterium transformation. We obtained a fluorescent strain for better observation of the infection process. Using two different inoculation methods of the fluorescent strain, we observed the traditional infection process through the body surface as well as through the digestive tract via feeding. Both infection modes can lead to larval death and mummification. Our findings demonstrated that during the artificial rearing of T. armoricanus, preventing C. farinosa pollution should be an important part of the disinfection of the rearing environment.

Prevalence and genotyping distribution of Enterocytozoon bieneusi in diarrheic pigs in Chongqing and Sichuan provinces, China.

The microsporidian fungal pathogen Enterocytozoon bieneusi is a unicellular parasite that infects humans and various animals, including pigs. Currently, there are few data on E. bieneusi infection a in diarrheic pigs in Chongqing and Sichuan Provinces, China. This study aims to determine the prevalence and genotype distribution of E. bieneusi in diarrheic pigs. In total, 514 fecal samples from diarrheic pigs were obtained from 14 large-scale farms in Chongqing and Sichuan Provinces (326 suckling pigs, 17 weaned pigs, 65 fattening pigs, and 106 sows). To identify the E. bieneusi genotypes, genomic DNA was isolated from the samples and tested by nested PCR, targeting the internal transcribed spacer region of the rRNA followed by DNA sequence analysis. The overall prevalence of E. bieneusi was 79.8% (410/514), with rates of 84.9% (90/106) in sows and 64.7% (11/17) in weaned pigs. We found 61 different genotypes, including seven known genotypes (E, F, CHG1, Peru8, CAF1, B, and BEB17) and 54 novel genotypes. These 54 new genotypes are variants of eight known genotypes (SDD2, A, B, HLJD-IV, PigSpEb1, O, JLD-I, and BEB17) based on their sequence similarities. Phylogenetically, all of the identified genotypes clustered with counterparts belonging to Group 1 and Group 2 of E. bieneusi. Therefore, we found a higher prevalence of E. bieneusi in sows than in preweaned and weaned pigs. These findings indicate that diarrheic pigs could be a potential reservoir host, which can contaminate the environment and be a source of microsporidia in humans and other animals.

Generation of Resistance to Nosema bombycis (Dissociodihaplophasida: Nosematidae) by Degrading NbSWP12 Using the Ubiquitin-Proteasome Pathway in Sf9-III Cells.

Nosema bombycis Naegeli (Dissociodihaplophasida: Nosematidae), an obligate intracellular parasite of the silkworm Bombyx mori, causes a devastating disease called pébrine. Every year pébrine will cause huge losses to the sericulture industry worldwide. Until now, there are no effective methods to inhibit the N. bombycis infection in silkworms. In this study, we first applied both the novel protein degradation Trim-Away technology and NSlmb (F-box domain-containing in the N-terminal part of supernumerary limbs from Drosophila melanogaster) to lepidopteran Sf9-III cells to check for specific degradation of a target protein in combination with a single-chain Fv fragment (scFv). Our results showed that the Trim-Away and NSlmb systems are both amenable to Sf9-III cells. We then created transgenic cell lines that overexpressed the protein degradation system and N. bombycis chimeric scFv targeting spore wall protein NbSWP12 and evaluated the effects of the insect transgenic cell lines on the proliferation of N. bombycis. Both methods could be applied to cell lines and both Trim-Away and NSlmb ubiquitin degradation systems effectively inhibited the proliferation of N. bombycis. Further, either of these degradation systems could be applied to individual silkworms through a transgenic platform, which would yield individual silkworms with high resistance to N. bombycis, thus greatly speeding up the process of acquiring resistant strains.

The NPC Families Mediate BmNPV Entry.

Baculovirus is a powerful tool for biological control in agriculture and foreign gene expression and delivery in insect and mammalian cells. Baculovirus enters host cells by multiple endocytic pathways; however, the current understanding of the Bombyx mori nucleopolyhedrovirus (BmNPV) entry mechanism remains limited. Previous studies have identified NPC1 and NPC2 as important host factors for viral infection in insect cells, although their exact role in viral infection has not yet been determined. In this study, we demonstrate that the BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 in Sf9 cells confers the virus the ability to enter into the nucleus of Sf9 cells. Additionally, the second luminal domain of BmNPC1 (BmNPC1-C) binds to the viral glycoprotein gp64, and preincubation of BmNPV with purified BmNPC1-C inhibits virus infection. Furthermore, knockout of the BmNPC2 protein results in reduced efficiency of viral fusion with the endosomal membrane, and BmNPC2 protein interacts directly with both viral envelope glycoprotein gp64 and the host BmNPC1 protein. BmNPC2 was found to be incorporated into progeny viral particles. Taken together, our results suggest that NPC2 protein incorporated into viral particles may facilitate viral infection through promoting the interaction of BmNPV and NPC1 in the endosome, thus enhancing viral fusion and escape from endosomes. These results, combined with those from previous studies, support that BmNPV hijacks two important cholesterol receptor members (NPC1 and NCP2) in the cholesterol intracellular transport pathway for viral entry into host cells. IMPORTANCE Baculovirus is an important biological factor for controlling insect populations and represents a powerful biological tool for gene delivery and expression. However, the host receptor of baculovirus is still unknown. In this study, we demonstrate that BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 confers the ability of virus to enter into the host cell nucleus in nonpermissive Sf9 cells. BmNPC2 can bind to the virus and promote progeny virion infection through the NPC1-NPC2 endosome cholesterol transport pathway. We believe that our study on the BmNPV entry mechanism will further facilitate the application of baculovirus systems in eukaryotic gene delivery. Not only can the cholesterol transport pathway NPC1 protein be used by a variety of enveloped viruses, but the NPC2 protein can also be used by viruses to infect host cells. This will provide new insights into the study of enveloped virus infection mechanisms.

Characterization of Pathway-Specific Regulator NigR for High Yield Production of Nigericin in Streptomyces malaysiensis F913.

Nigericin is a polyether antibiotic with potent antibacterial, antifungal, antimalarial and anticancer activity. NigR, the only regulator in the nigericin biosynthetic gene cluster in Streptomyces malaysiensis F913, was identified as a SARP family regulator. Disruption of nigR abolished nigericin biosynthesis, while complementation of nigR restored nigericin production, suggesting that NigR is an essential positive regulator for nigericin biosynthesis. Overexpression of nigR in Streptomyces malaysiensis led to significant increase in nigericin production compared to the wild-type strain. Nigericin production in the overexpression strain was found to reach 0.56 g/L, which may be the highest nigericin titer reported to date. Transcriptional analysis suggested that nigR is required for the transcription of structural genes in the nig gene cluster; quantitative RT-PCR analysis revealed that the expression of structural genes was upregulated in the nigR overexpression strain. Our study suggested that NigR acts in a positive manner to modulate nigericin production by activating transcription of structural genes and provides an effective strategy for scaling up nigericin production.

Utilization of Recombinant Baculovirus Expression System to Produce the RBD Domain of SARS-CoV-2 Spike Protein.

Continuous outbreaks of viral diseases in humans facilitates a need for the rapid development of viral test kits and vaccines. These require expression systems to produce a pure and high yield of target viral proteins. We utilized a baculovirus-silkworm expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. First, we had to develop a strategy for constructing a recombinant baculovirus for RBD expression. For this, the coding region of the Bombyx mori cypovirus (BmCPV) polyhedron was assembled with the Bombyx mori nuclear polyhedrosis virus (BmNPV) promoter. We demonstrated that the recombinant baculovirus has the ability to form polyhedrons within host silkworm cells. In addition, the encapsulated BVs are able to infect silkworms by ingestion and induce foreign protein expression. In this way, we utilized this novel system to obtain a high yield of the target foreign protein, the RBD of the SARS-CoV-2 S protein. However, the viral infection rate of our recombinant BV needs to be improved. Our study shed light on developing a highly efficient expression system for the production of antigens and subsequent immunoassays and vaccines.

Advances in the Genetic Manipulation of Nosema bombycis.

The microsporidium Nosema bombycis can infect and transmit both vertically and horizontally in multiple lepidopteran insects including silkworms and crop pests. While there have been several studies on the N. bombycis spore, there have been only limited studies on the N. bombycis sporoplasm. This chapter reviews what is known about this life cycle stage as well as published studies on purification of the N. bombycis sporoplasm and its survival in an in vitro cell culture system. Genetic transformation techniques have revolutionized the study of many pathogenic organisms. While progress has been made on the development of such systems for microsporidia, this critical problem has not been solved for these pathogens. This chapter provides a summary of the latest research progress on genetic manipulation of N. bombycis.