Targeting oncogenic KRAS with molecular brush-conjugated antisense oligonucleotides.

The mutant form of the guanosine triphosphatase (GTPase) KRAS is a key driver in human tumors but remains a challenging therapeutic target, making KRASMUT cancers a highly unmet clinical need. Here, we report a class of bottlebrush polyethylene glycol (PEG)-conjugated antisense oligonucleotides (ASOs) for potent in vivo KRAS depletion. Owing to their highly branched architecture, these molecular nanoconstructs suppress nearly all side effects associated with DNA-protein interactions and substantially enhance the pharmacological properties of the ASO, such as plasma pharmacokinetics and tumor uptake. Systemic delivery to mice bearing human non-small-cell lung carcinoma xenografts results in a significant reduction in both KRAS levels and tumor growth, and the antitumor performance well exceeds that of current popular ASO paradigms, such as chemically modified oligonucleotides and PEGylation using linear or slightly branched PEG. Importantly, these conjugates relax the requirement on the ASO chemistry, allowing unmodified, natural phosphodiester ASOs to achieve efficacy comparable to that of chemically modified ones. Both the bottlebrush polymer and its ASO conjugates appear to be safe and well tolerated in mice. Together, these data indicate that the molecular brush-ASO conjugate is a promising therapeutic platform for the treatment of KRAS-driven human cancers and warrant further preclinical and clinical development.

Online public concern about allergic rhinitis and its association with COVID-19 and air quality in China: an informative epidemiological study using Baidu index.

BACKGROUND: Allergic rhinitis is a common health concern that affects quality of life. This study aims to examine the online search trends of allergic rhinitis in China before and after the COVID-19 epidemic and to explore the association between the daily air quality and online search volumes of allergic rhinitis in Beijing. METHODS: We extracted the online search data of allergic rhinitis-related keywords from the Baidu index database from January 23, 2017 to June 23, 2022. We analyzed and compared the temporal distribution of online search behaviors across different themes of allergic rhinitis before and after the COVID-19 pandemic in mainland China, using the Baidu search index (BSI). We also obtained the air quality index (AQI) data in Beijing and assessed its correlation with daily BSIs of allergic rhinitis. RESULTS: The online search for allergic rhinitis in China showed significant seasonal variations, with two peaks each year in spring from March to May and autumn from August and October. The BSI of total allergic rhinitis-related searches increased gradually from 2017 to 2019, reaching a peak in April 2019, and declined after the COVID-19 pandemic, especially in the first half of 2020. The BSI for all allergic rhinitis themes was significantly lower after the COVID-19 pandemic than before (all p values < 0.05). The results also revealed that, in Beijing, there was a significant negative association between daily BSI and AQI for each allergic rhinitis theme during the original variant strain epidemic period and a significant positive correlation during the Omicron variant period. CONCLUSION: Both air quality and the interventions used for COVID-19 pandemic, including national and local quarantines and mask wearing behaviors, may have affected the incidence and public concern about allergic rhinitis in China. The online search trends can serve as a valuable tool for tracking real-time public concerns about allergic rhinitis. By complementing traditional disease monitoring systems of health departments, these search trends can also offer insights into the patterns of disease outbreaks. Additionally, they can provide references and suggestions regarding the public's knowledge demands related to allergic rhinitis, which can further be instrumental in developing targeted strategies to enhance population-based disease education on allergic diseases.

Sand supplementation favors tropical seagrass Thalassia hemprichii in eutrophic bay: implications for seagrass restoration and management.

BACKGROUND: Sediment is crucial for the unique marine angiosperm seagrass growth and successful restoration. Sediment modification induced by eutrophication also exacerbates seagrass decline and reduces plantation and transplantation survival rates. However, we lack information regarding the influence of sediment on seagrass photosynthesis and the metabolics, especially regarding the key secondary metabolic flavone. Meanwhile, sulfation of flavonoids in seagrass may mitigate sulfide intrusion, but limited evidence is available. RESULTS: We cultured the seagrass Thalassia hemprichii under controlled laboratory conditions in three sediment types by combining different ratios of in-situ eutrophic sediment and coarse beach sand. We examined the effects of beach sand mixed with natural eutrophic sediments on seagrass using photobiology, metabolomics and isotope labelling approaches. Seagrasses grown in eutrophic sediments mixed with beach sand exhibited significantly higher photosynthetic activity, with a larger relative maximum electron transport rate and minimum saturating irradiance. Simultaneously, considerably greater belowground amino acid and flavonoid concentrations were observed to counteract anoxic stress in eutrophic sediments without mixed beach sand. This led to more positive belowground stable sulfur isotope ratios in eutrophic sediments with a lower Eh. CONCLUSIONS: These results indicated that coarse beach sand indirectly enhanced photosynthesis in T. hemprichii by reducing sulfide intrusion with lower amino acid and flavonoid concentrations. This could explain why T. hemprichii often grows better on coarse sand substrates. Therefore, it is imperative to consider adding beach sand to sediments to improve the environmental conditions for seagrass and restore seagrass in eutrophic ecosystems.

Effects of steam explosion-modified rice bran dietary fiber on volatile flavor compounds retention and release of red date-flavored naan (ethnic specialty food of Xinjiang) during storage.

This study explored the effects of steam explosion-modified rice bran dietary fiber (S-RBDF) on red date-flavored naan quality and flavor characteristics. The results revealed that the rheological properties of the dough were improved with the incremental addition of S-RBDF (0-5%). The microstructure revealed that adding an appropriate amount of S-RBDF (1-5%) enabled more starch granules to be embedded in the dough network. Notably, the addition of 5% S-RBDF resulted in naan with an optimum specific volume and texture, which consumers preferred. Additionally, gas chromatography-mass spectrometry analysis showed that adding S-RBDF to naan contributed to the retention and sustained release of pleasant volatile compounds (e.g. red date flavor, etc.), while inhibiting the development of unpleasant volatile compounds by delaying the oxidation and decomposition of lipids and preserving the antioxidant phenolic compounds, thus contributing to flavor maintenance of naan during storage. Overall, these results provided a foundation for developing high-quality flavored naan.

Chinese surgical robot-assisted surgery for parotid tumor: a case report.

Robotic surgery is known as the "third technological revolution" in the field of surgery, and is an important milestone in the development of modern surgery. However, our country's innovative surgical robot industry is still in its early stages, and it is only being utilized in certain surgical fields. To explore the effectiveness of the application of domestic surgical robot in oral and maxillofacial surgery, the author successfully completed a case of benign parotid tumor resection with the assistance of a domestic autonomous robot. The operation was successful, facial nerve function was preserved, and postoperative wound healing was good.

Evaluation of a newly developed oral and maxillofacial surgical robotic platform (KD-SR-01) in head and neck surgery: a preclinical trial in porcine models.

Traditional open head and neck surgery often leaves permanent scars, significantly affecting appearance. The emergence of surgical robots has introduced a new era for minimally invasive surgery. However, the complex anatomy of the head and neck region, particularly the oral and maxillofacial areas, combined with the high costs associated with established systems such as the da Vinci, has limited the widespread adoption of surgical robots in this field. Recently, surgical robotic platform in China has developed rapidly, exemplified by the promise shown by the KangDuo Surgical Robot (KD-SR). Although the KD-SR has achieved some results comparable to the da Vinci surgical robot in urology and colorectal surgery, its performance in complex head and neck regions remains untested. This study evaluated the feasibility, effectiveness, and safety of the newly developed KD-SR-01, comparing it with standard endoscopic systems in head and neck procedures on porcine models. We performed parotidectomy, submandibular gland resection, and neck dissection, collected baseline characteristics, perioperative data, and specifically assessed cognitive workload using the NASA-TLX. None of the robotic procedures were converted to endoscopic or open surgery. The results showed no significant difference in operation time between the two groups (P = 0.126), better intraoperative bleeding control (P = 0.001), and a significant reduction in cognitive workload (P < 0.001) in the robotic group. In conclusion, the KD-SR-01 is feasible, effective, and safe for head and neck surgery. Further investigation through well-designed clinical trials with long-term follow-up is necessary to establish the full potential of this emerging robotic platform.

[Fatty acid composition of edible marine fish in Zhoushan, Zhejiang province].

OBJECTIVE: To analyze the main fatty acids in edible marine fish from Zhoushan, Zhejiang province. METHODS: From September to October 2011, a total of 186 edible marine fish (31 species,6 individual fishes/species) were collected in local markets. Total lipids of edible part were extracted by Folch's method and fatty acids were separated and quantified by gas chromatographic after the homogenization of edible part. The differences of composition of n-6 polyunsaturated fatty acid (n-6 PUFA), n-3 polyunsaturated fatty acid (n-3 PUFA),saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) among fishes were analyzed. RESULTS: Among the 31 fishes, total lipids were highest in Auxis thazard ((13.2 ± 1.2)g/100 g edible part) and lowest in Thamnaconus modestus ((0.6 ± 0.1)g/100 g edible part). Total n-6 PUFA were highest in Mugil cephalus ((875.7 ± 506.4)mg/100 g edible part) and lowest in Seriola quinqueradiata((2.1 ± 1.9)mg/100 g edible part). Total n-3 PUFA were highest in Auxis thazard ((2623.8 ± 426.1)mg/100 g edible part) and lowest in Scoliodon sorrakowah ((82.0 ± 13.9)mg/100 g edible part). SFA were highest in Trachinotus ovatus((3014.9 ± 379.0)mg/100 g edible part) and lowest in Seriola quinqueradiata ((89.7 ± 5.8)mg/100 g edible part). MUFA were highest in Coilia nasus ((3335.7 ± 383.5)mg/100 g edible part) and lowest in Thamnaconus modestus ((32.1 ± 16.9)mg/100 g edible part). CONCLUSION: There were significant differences of composition of total lipids and of fatty acids among 31 edible marine fish species from Zhoushan.

Did the COVID-19 pandemic impact urticaria information-seeking behavior in China? A retrospective longitudinal study.

Purpose: To investigate information-seeking behavior related to urticaria before and during the COVID-19 pandemic in China. Methods: Search query data for terms related to urticaria were retrieved using Baidu Index database from October 23, 2017 to April 23, 2022, and daily COVID-19 vaccination doses data were obtained from the website of the Chinese Center for Disease Control and Prevention. Among the 23 eligible urticaria search terms, four urticaria themes were generated as classification, symptom, etiology, and treatment of urticarial, respectively. Baidu Search Index (BSI) value for each term were extracted to analyze and compare the spatial and temporal distribution of online search behavior for urticaria before and after the COVID-19 pandemic, and to also explore the correlation between search query and daily COVID-19 vaccination doses. Results: The classification of urticaria accounted for nearly half of the urticaria queries on the internet. Regular seasonal patterns of BSI were observed in urticaria-related online search, by attaining its highest level in spring and summer and lowest level in winter. The BSIs of all urticaria themes significantly increased after the COVID-19 pandemic than that before the pandemic (all P<0.05). Xizang, Qinghai and Ningxia are the most active geographical areas for increased urticaria-searching activities after the COVID-19 pandemic. There was also a significant positive correlation between daily BSIs and daily COVID-19 vaccination doses in each urticaria theme. Cross-correlation analysis found that the search of symptom, etiology, and treatment attained their strongest correlation with daily COVID-19 vaccination doses at 11-27 days before the injection of vaccine, imply vaccination hesitation related to concerns of urticaria. Conclusions: This study used the internet as a proxy to provide evidence of public search interest and spatiotemporal characteristics of urticaria, and revealed that the search behavior of urticaria have increased significantly after the COVID-19 pandemic and COVID-19 vaccination. It is anticipated that the findings about such increase in search behavior, as well as the behavior of urticaria-related vaccine-hesitancy, will help guide public health education and policy regulation.

Effects of herbivore on seagrass, epiphyte and sediment carbon sequestration in tropical seagrass bed.

Herbivores strongly affect the ecological structure and functioning in seagrass bed ecosystems, but may exhibit density-dependent effects on primary producers and carbon sequestration. This study examined the effects of herbivorous snail (Cerithidea rhizophorarum) density on snail intraspecific competition and diet, dominant seagrass (Thalassia hemprichii) and epiphyte growth metrics, and sediment organic carbon (SOC). The growth rates of the herbivorous snail under low density (421 ind m-2) and mid density (842 ind m-2) were almost two times of those at extremely high density (1684 ind m-2), indicating strong intraspecific competition at high density. Herbivorous snails markedly reduced the epiphyte biomass on seagrass leaves. Additionally, the seagrass contribution to herbivorous snail as food source under high density was about 1.5 times of that under low density, while the epiphyte contribution under low density was 3 times of that under high density. A moderate density of herbivorous snails enhanced leaf length, carbon, nitrogen, total phenol and flavonoid contents of seagrasses, as well as surface SOC content and activities of polyphenol oxidase and ß-glucosidase. However, high density of herbivorous snails decreased leaf glucose, fructose, detritus carbon, and total phenols contents of seagrasses, as well as surface SOC content and activities of polyphenol oxidase and ß-glucosidase. Therefore, the effects of herbivorous snail on seagrass, epiphyte and SOC were density-dependent, and moderate density of herbivorous snail could be beneficial for seagrasses to increase productivity. This provided theoretical guidance for enhancing carbon sink in seagrass bed and its better conservation.

A Kano model-based demand analysis and perceived barriers of pulmonary rehabilitation interventions for patients with chronic obstructive pulmonary disease in China.

BACKGROUND: Pulmonary rehabilitation (PR) has been recognized to be an effective therapy for chronic obstructive pulmonary disease (COPD). However, in China, the application of PR interventions is still less promoted. Therefore, this cross-sectional study aimed to understand COPD patients' intention to receive PR, capture the potential personal, social and environmental barriers preventing their willingness of receiving PR, and eventually identify demanding PR services with the highest priority from patients' point of view. METHODS: In total 237 COPD patients were recruited from 8 health care facilities in Zhejiang, China. A self-designed questionnaire was applied to investigate patients' intention to participate in PR and potentially associated factors, including personal dimension such as personal awareness, demographic factors, COPD status and health-related literacy/behaviors, as well as social policies and perceived environmental barriers. The demand questionnaire of PR interventions based on the Kano model was further adopted. RESULTS: Among the 237 COPD patients, 75.1% of COPD patients were willing to participate in PR interventions, while only 62.9% of the investigated patients had heard of PR interventions. Over 90% of patients believed that the cost of PR services and the ratio of medical insurance reimbursement were potential obstacles hindering them from accepting PR services. The multiple linear regression analysis indicated that the PR skills of medical staff, knowledge promotion and public education levels of PR in the community, patients' transportation concerns and degree of support from family and friends were significantly associated with willingness of participation in PR interventions. By using the Kano model, the top 9 most-requisite PR services (i.e., one-dimensional qualities) were identified from patients' point of view, which are mainly diet guidance, education interventions, psychological interventions and lower limb exercise interventions. Subgroup analysis also revealed that patients' demographics, such as breathlessness level, age, education and income levels, could influence their choice of priorities for PR services, especially services related to exercise interventions, respiratory muscle training, oxygen therapy and expectoration. CONCLUSIONS: This study suggested that PR-related knowledge education among patients and their family, as well as providing basic package of PR services with the most-requisite PR items to COPD patients, were considerable approaches to promote PR attendance in the future.

Response of tropical seagrass palatability based on nutritional quality, chemical deterrents and physical defence to ammonium stress and its subsequent effect on herbivory.

Seagrass-herbivore interactions play a principal role in regulating the structure and function of coastal food webs, which were affected by nutrient enrichment. Seawater nutrient enrichment might change seagrass palatability by altering seagrass physical and chemical traits, consequently modulating herbivory patterns, but this remains elusive. In this study, the dominant tropical seagrass Thalassia hemprichii was cultured in different ammonium concentrations to examine the response of seagrass nutritional quality, deterrent secondary metabolites, and leaf toughness, as well as the subsequent effect of the changed physical (e.g., leaf toughness) and chemical traits (e.g., nitrogen content; total phenol) on the grazing activity of the herbivorous snail Cerithidea rhizophorarum. Ammonium enrichment enhanced seagrass nutritional quality and decreased physical defence. Low ammonium enrichment increased total phenol content, while high ammonium enrichment reduced it. Both low and high ammonium enrichment enhanced the grazing intensity of C. rhizophorarum on seagrass. Interestingly, nutritional quality mostly determined the herbivory preference of C. rhizophorarum on the intact seagrass having physical structure, with a chemical deterrent (total phenol) playing a secondary role. In contrast, chemical deterrent mainly determined the grazing intensity on agar seagrass food which was made artificially to exclude physical structure. This indicated that seagrass leaf physical structure might hinder phenol compounds from deterring herbivores. Overall, the results presented here demonstrate that ammonium enrichment remarkably increased seagrass palatability and subsequently induced higher susceptibility to herbivory, which might induce seagrass loss.

Heavy metal accumulation and ecological risk on four seagrass species in South China.

Zn, Pb, Cr, Cu, Ni, Cd concentration and ecological risk were studied in three bays to evaluate the heavy metal (HM) contamination of seagrasses. Seasonal HM accumulation varied according to locations, seagrass species and tissues. Halophila beccarii had much higher HM concentrations except for Cr in Zhelin Bay, however, bioconcentration factors (BCF) of Cu, Ni, Pb, Zn were higher in Liusha than Zhelin Bay. Cr was much enriched in Thalassia hemperichii and Enhalus acoroides than Halophila beccarii and Halophila ovalis. Cr, Cu, Ni, Pb were easy to accumulate in belowground tissues in Halophila ovalis. In contrast, almost all HM were more enriched in aboveground tissues in other species. Generally, BCF exceeding 1 and high metal pollution index suggested HM had potential ecological risk on seagrasses. The results provide the reference for managing and protecting seagrass ecosystem in South China, and are significant to expand the global seagrass detection network.

Low Light Availability Reduces the Subsurface Sediment Carbon Content in Halophila beccarii From the South China Sea.

Eutrophication, dredging, agricultural and urban runoffs, and epiphyte overgrowth could reduce light availability for seagrass. This may affect "blue carbon" stocks in seagrass beds. However, little research is available on the effect of light intensities on carbon sequestration capacity in seagrass beds, especially small-bodied seagrasses. The dominant seagrass Halophila beccarii, a vulnerable species on the IUCN Red List, was cultured in different light intensities to examine the response of vegetation and sediment carbon in seagrass beds. The results showed that low light significantly reduced leaf length and above-ground biomass, while carbon content in both above-ground and below-ground tissues were not affected. Low light reduced both the above-ground biomass carbon and the total biomass carbon. Interestingly, while under saturating light conditions, the subsurface and surface carbon content was similar, under low light conditions, subsurface sediment carbon was significantly lower than the surface content. The reduction of subsurface sediment carbon might be caused by less release flux of dissolved organic carbon from roots in low light. Taken together, these results indicate that reduced light intensities, to which these meadows are exposed to, will reduce carbon sequestration capacity in seagrass beds. Measures should be taken to eliminate the input of nutrients on seagrass meadows and dredging activities to maintain the "blue carbon" storage service by enhancing light penetration into seagrass.

Interspecific differences in root exudation for three tropical seagrasses and sediment pore-water dissolved organic carbon beneath them.

Seagrass beds act as blue carbon sinks globally; however, little attention has been given to carbon dynamics in the seagrass rhizosphere. Hence, in this study, the quantity and characteristics of dissolved organic carbon (DOC) from root exudation of the three dominant tropical seagrasses (Thalassia hemprichii, Enhalus acoroides, and Cymodocea rotundata) and sediment pore water beneath them were compared, to examine their interspecific differences, and to establish a connection between seagrass root exudation and sediment carbon. The rate of root-exuded DOC from T. hemprichii (2.15 ± 1.06 mg g DW root-1 h-1) was significantly higher (p < 0.05) than that from E. acoroides (0.72 ± 0.39 mg g DW root-1 h-1) and C. rotundata (0.46 ± 0.25 mg g DW root-1 h-1). Root exudation rates were more affected by root hair density and root hair length than by root carbon, nitrogen, and soluble sugar content. Simultaneously, DOC concentrations of the sediment pore water beneath T. hemprichii, E. acoroides and C. rotundata were 22.05 ± 11.61 mg l-1, 15.55 ± 2. 66 mg l-1, and 14.32 ± 1.82 mg l-1, respectively. The corresponding absorption coefficients at 254 nm (a254) were 30.53 ± 18.00, 17.31 ± 2.24, and 14.07 ± 2.03, respectively, while the relevant specific ultraviolet absorbances at 254 nm (SUVA254) were 1.38 ± 0.29, 1.19 ± 0.26 and 1.03 ± 0.28, respectively. Therefore, the roots of T. hemprichii exuded DOC at a higher rate, leading to a higher pore-water DOC pool in the sediment. This suggests that T. hemprichii played a greater role in the sediment carbon pool through root exudation. Thus, it can be considered as the priority species for transplantation to promote the carbon sink function of seagrass beds.

SARS-CoV-2 viral shedding characteristics and potential evidence for the priority for faecal specimen testing in diagnosis.

This study aimed to identify the specimen type that has high positivity and its proper sampling time for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing to promote diagnostic efficiency. All SARS-CoV-2-infected patients with a laboratory-confirmed diagnosis in Zhoushan City were followed up for viral shedding in respiratory tract specimens and faecal samples. Positivity was analysed both qualitatively and quantitatively by proper statistical approaches with strong testing power. Viral shedding in respiratory tract and faecal specimens was prolonged to 45 and 40 days after the last exposure, respectively. The overall positive rate in respiratory tract specimens was low and relatively unstable, being higher in the early-to-mid stage than in the mid-to-late stage of the disease course. Compared with respiratory tract specimens, faecal samples had a higher viral load, higher overall positive rate, and more stable positivity in different disease courses and varied symptomatic status. Faecal specimens have the potential ability to surpass respiratory tract specimens in virus detection. Testing of faecal specimens in diagnosis, especially for identifying asymptomatic carriers, is recommended. Simultaneously, testing respiratory tract specimens at the early-to-mid stage is better than testing at the mid-to-late stage of the disease course. A relatively small sample size was noted, and statistical approaches were used to address it. Information was missing for both specimen types at different stages of the disease course due to censored data. Our research extends the observed viral shedding in both specimen types and highlights the importance of faecal specimen testing in SARS-CoV-2 diagnosis. Healthcare workers, patients, and the general public may all benefit from our study findings. Disposal of sewage from hospitals and residential areas should be performed cautiously because the virus sheds in faeces and can last for a long time.

Analysis of N-Terminal Nucleotide Sequence of NDV Fusion Protein.

Fusion protein of Newcastle disease virus is a glycoprotein with antigenicity. The cDNA of fusion gene of NDV strain F48E8 was cloned by RT-PCR, and the sequence of F48E8 was compared with other two virulent strains, Australia-victoria and Italien. Results show that the sequences of N-terminus are very similar, and cysteine residues and the potential glycosylation sites are relatively conserved. However, there are a few differences in the region of signal peptides.

Sequence Analysis of SOD Gene of Bombyx mori Nuclear Polyhedrosis Virus and Its Expression in E.coli.

Superoxide dismutases scavenge superoxide radicals and protect cells from oxidative stress. SOD gene of Bombyx mori nuclear polyhedrosis virus has been cloned by PCR, and is expressed in E.coli with the activity of SOD being 576.13 u/mL. DNA sequence analysis shows that SOD gene of BmNPV encodes 151 amino acids. The homology between BmNPV and AcNPV of the nucleotide sequences of SOD gene is 97.2%, and 56% between BmNPV and human SOD1.

Functional Analysis of Helicase Gene Promoter and Homologous Region 3 Enhancer in Bombyx mori Nuclear Polyhedrosis Virus.

The promoter of the helicase gene, including 510 bp upstream of ATG,was cloned and sequenced, and was found that it had both early and late RNA initiation sites. The initiation codon ATG was deleted by using point mutation. Luciferase gene, as a reporter gene, was fused with the promoter region to construct the plsmid pBm hel 510 luc. When pBm hel 510 luc was transfected into Bm-5 and Sf-21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and transactivated by viral factors. Baculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to alter the rate of transcription for cis-linked promoters. BmNPV hr3 was cloned into a downstream site of luc gene, to study the effect of this enhancer on hel 510 promoter activity. The transient expression in transfected insect cell lines and silkworm larvae indicated that hr 3 could enhance the transcriptional level of hel 510 promoter by about 7 000 and 1 000 fold, respectively.

[Identification of functional region of helicase gene promoter in Bombyx mori nuclear polyhedrosis virus].

DNA helicases are essential for replication of baculoviruses. It was found that the helicase gene promoter of Bombyx mori nuclear polyhedrosis virus, including 510 bp upstream of ATG, had both early and late RNA initiation sites and could be recognized by cellular RNA polymerase. Transient expression assays in uninfected Sf-21 cells indicated that the helicase gene promoter could be classified as a delayed-early gene promoter. Deletion analysis by PCR showed that the regulation region of its basic transcription was mainly within -510 to -410 bp upstream of ATG. However, the basic activity was still detected with a deletion to -98 bp relative to ATG. In the presence of viral factors, deletion between -510 to -410 bp relative to ATG did not significantly reduce the promoter activity compared to the full-length promoter (510 bp). The remarkable reduction in the promoter activity was observed with continuous deletions. It suggests, therefore, that cis-acting elements responsive to viral factors are mainly located within the range of -410 to -309 bp upstream of ATG.

Expression of the Arrowhead Proteinase Inhibitor B (API) Gene in the Silkworm Larvae and Pupae.

The arrowhead proteinase inhibitor B (API) gene was cloned into baculovirus transfer vectors of pBacPAK8 and pOSX, and two recombinant transfer vectors, pBacPAK (API) and pOSX (API), were constructed. Co-infection was accomplished with either one of the recombinant transfer vectors and Bm-BacPAK6 DNA in BmN cells. Then recombinant virus of CrBK9, CrBK10 and BX was selected. API gene was successfully expressed in silkworm larvae and pupae. The recombinant API (rAPI) showed strong inhibitive activity on bovine pancreas trypsin. The inhibiting activity of the expression product was 1.0x10(5) u/ml haemolymph for silkworm larvae and 1.3x10(5)u/ml haemolymph for pupae. In addition, SDS-PAGE of the preliminarily purified rAPI showed its molecular weight to be about 19 kD.