Combining slow-release fertilizer and plastic film mulching reduced the carbon footprint and enhanced maize yield on the Loess Plateau.

Agricultural practices significantly contribute to greenhouse gas (GHG) emissions, necessitating cleaner production technologies to reduce environmental pressure and achieve sustainable maize production. Plastic film mulching is commonly used in the Loess Plateau region. Incorporating slow-release fertilizers as a replacement for urea within this practice can reduce nitrogen losses and enhance crop productivity. Combining these techniques represents a novel agricultural approach in semi-arid areas. However, the impact of this integration on soil carbon storage (SOCS), carbon footprint (CF), and economic benefits has received limited research attention. Therefore, we conducted an eight-year study (2015-2022) in the semi-arid northwestern region to quantify the effects of four treatments [urea supplied without plastic film mulching (CK-U), slow-release fertilizer supplied without plastic film mulching (CK-S), urea supplied with plastic film mulching (PM-U), and slow-release fertilizer supplied with plastic film mulching (PM-S)] on soil fertility, economic and environmental benefits. The results revealed that nitrogen fertilizer was the primary contributor to total GHG emissions (≥71.97%). Compared to other treatments, PM-S increased average grain yield by 12.01%-37.89%, water use efficiency by 9.19%-23.33%, nitrogen accumulation by 27.07%-66.19%, and net return by 6.21%-29.57%. Furthermore, PM-S decreased CF by 12.87%-44.31% and CF per net return by 14.25%-41.16%. After eight years, PM-S increased SOCS (0-40 cm) by 2.46%, while PM-U decreased it by 7.09%. These findings highlight the positive effects of PM-S on surface soil fertility, economic gains, and environmental benefits in spring maize production on the Loess Plateau, underscoring its potential for widespread adoption and application.

Enhanced antibiofilm potential of low-intensity pulsed ultrasound combined with 0.35% povidone-iodine in a rat model of periprosthetic joint infection.

Aims: Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). Methods: A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR. Results: The group with LIPUS and 0.35% PI exhibited decreased levels of serum biochemical markers, improved weightbearing scores, reduced reactive bone changes, absence of viable bacteria, and decreased inflammation compared to the Control group. Despite the greater antibiofilm activity observed in the PI group compared to the LIPUS and saline group, none of the monotherapies were successful in preventing reactive bone changes or eliminating the infection. Conclusion: In the rat model of PJI treated with DAIR, LIPUS combined with 0.35% PI demonstrated stronger antibiofilm potential than monotherapy, without impairing any local soft-tissue.

Straw returning and nitrogen reduction: Strategies for sustainable maize production in the dryland.

Implementing continue straw returning practices and optimizing nitrogen application can mitigate nitrogen losses and enhance nitrogen use efficiency (NUE) in dryland. 15N-labeled technique offers a robust approach for tracking fertilizer nitrogen fate and assessing nitrogen use efficiency. Based on the continue (>6 yr) experiment, we conducted a two-year experiment (2020 and 2021) to evaluate the effects of straw returning and nitrogen management under plastic film mulching on 15N recovery rates, N2O emissions and maize yield with three treatments: no straw returning with 225 kg N·ha-1 under plastic film mulching (RP-N225), straw returning with 225 kg N·ha-1 under plastic film mulching (RPS-N225), and straw returning with 20% nitrogen reduction (180 kg N·ha-1) under plastic film mulching (RPS-N180). After six years, both continue straw returning with plastic film mulching increased uptake of fertilizer nitrogen, had higher 15N recovery rates than RP-N225, leading to increased 15N accumulation in grain and aboveground biomass, ultimately enhancing yield. The RPS-N225 treatment exhibited the highest spring maize yield and nitrogen harvest index. The RPS-N180 treatment significantly increased maize yield more than RP-N225 and had the highest NUE, partial factor productivity of nitrogen fertilizer, and nitrogen uptake efficiency, with improvements ranging from 1.7 to 2.4%, 19.3-29.6%, and 17.3-27.5%, respectively, compared to the other treatments. Moreover, RPS-N225 resulted in significantly higher cumulative N2O emissions and yield-scaled N2O emissions than the other treatments, whereas the RPS-N180 treatment significantly decreased yield-scaled N2O emissions compared to RP-N225. Hence, combining continue straw returning with appropriate nitrogen reduction can effectively increase maize yield and yield-scaled N2O emissions. By offering insights into optimizing nitrogen fertilizer management after continue maize straw return, this study is contributed to widespread adoption of straw return practices and sustainable agricultural development in semi-arid areas.

Integrated monitoring and prediction of thermal discharge from nuclear power plants using satellite, UAV, and numerical simulation.

Global nuclear power is surging ahead in its quest for global carbon neutrality, eyeing an anticipated installed capacity of 436 GW for coastal nuclear power plants by 2040. As these plants operate, they emit substantial amounts of warm water into the ocean, known as thermal discharge, to regulate the temperature of their nuclear reactors. This discharge has the potential to elevate the temperature of the surrounding seawater, potentially influencing the marine ecosystem in the discharge vicinity. Therefore, our study area is on the Qinshan and Jinqimen Nuclear Power Plants in China, employing a blend of Landsat 8/9, and unmanned aerial vehicle (UAV) imagery to gather sea surface temperature (SST) data. In situ measurements validate the temperature data procured through remote sensing. Leveraging these SST observations alongside hydrodynamic and meteorological data from field measurements, we input them into the MIKE 3 model to prognosticate the three-dimensional (3D) spatial distribution and temperature elevation resulting from thermal discharge. The findings reveal that (1) satellite remote sensing can instantly acquire the horizontal distribution of thermal discharge, but with a spatial resolution much lower than that of UAV. The spatial resolution of UAV is higher, but the imaging efficiency of UAV is only 1/40,000 of that of satellite remote sensing. (2) Numerical simulation models can predict the 3D spatial distribution of thermal discharge. Although UAV and satellite remote sensing cannot directly obtain the 3D spatial distribution of thermal discharge, using remotely sensed SST as the temperature field input for the MIKE 3 model can reduce the quantity of measured temperature data and lower the cost of numerical simulation. (3) In the process of monitoring and predicting the thermal discharge of nuclear power plants, achieving an effective balance between monitoring accuracy and cost can be realized by comprehensively considering the advantages and costs of satellite, UAV, and numerical simulation technologies.

Evaluation of vaccine candidates against Rhodococcus equi in BALB/c mice infection model: cellular and humoral immune responses.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that mainly causes fatal lung and extrapulmonary abscesses in foals and immunocompromised individuals. To date, no commercial vaccine against R. equi exists. We previously screened all potential vaccine candidates from the complete genome of R. equi using a reverse vaccinology approach. Five of these candidates, namely ABC transporter substrate-binding protein (ABC transporter), penicillin-binding protein 2 (PBD2), NlpC/P60 family protein (NlpC/P60), esterase family protein (Esterase), and M23 family metallopeptidase (M23) were selected for the evaluation of immunogenicity and immunoprotective effects in BALB/c mice model challenged with R. equi. The results showed that all five vaccine candidate-immunized mice experienced a significant increase in spleen antigen-specific IFN-γ- and TNF-α-positive CD4 + and CD8 + T lymphocytes and generated robust Th1- and Th2-type immune responses and antibody responses. Two weeks after the R. equi challenge, immunization with the five vaccine candidates reduced the bacterial load in the lungs and improved the pathological damage to the lungs and livers compared with those in the control group. NlpC/P60, Esterase, and M23 were more effective than the ABC transporter and PBD2 in inducing protective immunity against R. equi challenge in mice. In addition, these vaccine candidates have the potential to induce T lymphocyte memory immune responses in mice. In summary, these antigens are effective candidates for the development of protective vaccines against R. equi. The R. equi antigen library has been expanded and provides new ideas for the development of multivalent vaccines.

Monitoring of temperature rise in global nuclear power plant thermal discharge from 2013 to 2022.

The development of nuclear power plants is progressing rapidly worldwide. However, there is currently a lack of dynamic monitoring of the thermal discharge temperature rise from these plants, making it unclear to governments where their nuclear power thermal discharges stand globally. We hypothesize that between 2013 and 2022, there are significant temporal and spatial differences in the thermal discharge temperature rise from nuclear power plants globally. Temporal differences are expected to reflect a country's nuclear power installed capacity and thermal discharge treatment capabilities, while spatial differences are related to the type of water bodies where nuclear power plants are located. To test these hypotheses, we utilized Landsat data to get the distribution range of thermal discharge and temperature rise levels ranging from 1 °C to 4 °C, and compared the temporal and spatial characteristics of temperature rise in different countries. The results indicate that: (1) Currently, China, the United States, and Canada rank among the top three globally in terms of the area experiencing temperature rise due to thermal discharge, which correlates with the total installed capacity of nuclear power in these countries. (2) Countries such as Russia, Finland, and Mexico exhibit larger areas with a 4 °C temperature rise level per unit installed capacity, with their thermal rise area per unit installed capacity (TRAUIC) exceeding the global average by more than 1.5 times. (3) The spatial dispersion trends of thermal discharges from nuclear power plants vary across different types of water bodies. For nuclear power plants located in bays, thermal discharges primarily disperse along the coast, while in open sea and lakes, thermal discharges tend to spread in a fan-shaped pattern. The findings of this study are crucial for understanding the efficiency of thermal discharge from nuclear power plants across different countries globally, assessing potential environmental risks during the operation of these plants, and promoting the safe and orderly development of nuclear power plants worldwide.

Negatively charged nanodiscs for the reduction of toxicity and enhanced efficacy of polymyxin B against Acinetobacter baumannii sepsis.

The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. STATEMENT OF SIGNIFICANCE: Multidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii, currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.

Finotonlimab with chemotherapy in recurrent or metastatic head and neck cancer: a randomized phase 3 trial.

Immunotherapy combined with chemotherapy regimen has been shown to be effective in recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). However, due to the small number of patients, its efficacy remains controversial in Asian populations, particularly in mainland China. Here a randomized, double-blind phase 3 trial evaluated the efficacy and safety of finotonlimab (SCT-I10A), a programmed cell death 1 (PD-1) monoclonal antibody, combined with cisplatin plus 5-fluorouracil (C5F) for the first-line treatment of R/M HNSCC. Eligible patients (n = 370) were randomly 2:1 assigned to receive finotonlimab plus C5F (n = 247) or placebo plus C5F (n = 123). The primary endpoint was overall survival (OS). In the finotonlimab plus C5F group, OS was 14.1 months (95% confidence interval (CI) 11.1-16.4), compared with 10.5 months (95% CI 8.1-11.8) in the placebo plus C5F group. The hazard ratio was 0.73 (95% CI 0.57-0.95, P = 0.0165), meeting the predefined superiority criteria for the primary endpoint. Finotonlimab plus C5F showed significant OS superiority compared with C5F alone and acceptable safety profile with R/M HNSCC, supporting its use as a first-line treatment option for R/M HNSCC. These results validate the efficacy and safety of the combination of finotonlimab and C5F in Asian patients with R/M HNSCC. ClinicalTrials.gov identifier: NCT04146402 .

ISAba1-mediated intrinsic chromosomal oxacillinase amplification confers carbapenem resistance in Acinetobacter baumannii.

Tandem amplification of carbapenemase genes increases gene copy number and enhances carbapenem resistance. These amplifications are often heterogeneous, transient, and located on plasmids, which also contribute to heteroresistance. Amplification of encoding genes is especially important for enzymes with low hydrolysis activity, which are often overlooked. Here, we reported an intrinsic oxacillinase oxaAb amplification flanked by ISAba1. The amplification is in the chromosome and contains up to 25 repeats. We provided genomic, transcriptomic, and proteomic evidence that the amplification resulted in oxacillinase overproduction. Notably, no point mutations of oxaAb were found during the amplification process. Strains of Acinetobacter baumannii with intrinsic amplified or external transformed ISAba1-oxaAb exhibited higher meropenem hydrolysis activity. Furthermore, the number of repeats in the amplification decreased gradually over a period of 21 d cultured with carbapenem withdrawal. However, upon re-exposure to meropenem, the ISAba1 flanked oxaAb responded rapidly, with repeat numbers reaching or exceeding pre-carbapenem withdrawal levels within 24 h. Taken together, these findings suggest that ISAba1-mediated gene amplification and overproduction of intrinsic low-activity oxacillinase oxaAb resulted in carbapenem resistance.

Discovery of Sophoridine α-Aryl Propionamide Derivative ZM600 as a Novel Antihepatic Fibrosis Agent.

Hepatic stellate cells (HSCs) activation is a key event in the development of liver fibrosis, and blockage of the activation of HSCs has been shown to alleviate liver fibrosis. Sophoridine, a bioactive alkaloid found in many Chinese herbs, exhibits a broad spectrum of pharmacological effects, but its activities are not strong. In this study, a series of structurally modified derivatives of sophoridine were designed and synthesized. Among them, sophoridine α-aryl propionamide derivative ZM600 displayed a significant inhibitory effect on the activation of HSCs. The in vivo experiment demonstrated that ZM600 markedly ameliorated carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced liver fibrosis with a significant improvement of extracellular matrix deposition. Mechanism investigations revealed that ZM600 specifically inhibited the activation of NF-κB, PI-3K/AKT, and TGF-ß/Smads signaling pathways. These results suggest that ZM600 has a protective effect on liver fibrosis, which provides a new candidate for the treatment of liver fibrosis.

[Effects of tramadol hydrochloride preemptive analgesia in kyphoplasty of thoracolumbar osteoporotic fractures under local anesthesia].

OBJECTIVE: To explore preemptive analgesic effect of preoperative intramural tramadol injection in percutaneous kyphoplasty (PKP) of vertebrae following local anesthesia. METHODS: From August 2019 to June 2021, 118 patients with thoraco lumbar osteoporotic fractures were treated and divided into observation group and control group, with 59 patients in each gruop. In observation group, there were 26 males and 33 females, aged from 57 to 80 years old with an average of (67.69±4.75)years old;14 patients on T11, 12 patients on T12, 18 patients on L1, 15 patients on L2;tramadol with 100 mg was injected intramuscularly half an hour before surgery in observation group. In control group, there were 24 males and 35 females, aged from 55 to 77 years old with an average of (68.00±4.43) years old;19 patients on T11, 11 patients on T12, 17patients on L1, 12 patients on L2;the same amount of normal saline was injected intramuscularly in control group. Observation indicators included operation time, intraoperative bleeding, visual analogue scale (VAS) evaluation and recording of preoperative (T0), intraoperative puncture(T1), and working cannula placement (T2) between two groups of patients, at the time of balloon dilation (T3), when the bone cement was injected into the vertebral body (T4), 2 hours after the operation (T5), and the pain degree at the time of discharge(T6);adverse reactions such as dizziness, nausea and vomiting were observed and recorded;the record the patient's acceptance of repeat PKP surgery. RESULTS: All patients were successfully completed PKP via bilateral pedicle approach, and no intravenous sedative and analgesic drugs were used during the operation. There was no significant difference in preoperative general data and VAS(T0) between two groups (P>0.05). There was no significant difference in operation time and intraoperative blood loss between the two groups (P>0.05). VAS of T1, T2, T3, T4 and T5 in observation group were all lower than those in control group(P<0.05), and there was no significant difference in T6 VAS (P>0.05). T6 VAS between two groups were significantly lower than those of T0, and the difference was statistically significant (P<0.05). There was no significant difference in incidence of total adverse reactions between two groups (P>0.05). There was a statistically significant difference in the acceptance of repeat PKP surgery (P<0.05). CONCLUSION: Half an hour before operation, intramuscular injection of tramadol has a clear preemptive analgesic effect for PKP of single-segment thoracolumbar osteoporotic fracture vertebral body under local anesthesia, which could increase the comfort of patients during operation and 2 hours after operation, and improve patients satisfaction with surgery.

CircRNA SEC24A promotes osteoarthritis through miR-107-5p/CASP3 axis.

Background: Osteoarthritis (OA) is the most frequently diagnosed chronic joint disease. CircSEC24A is significantly elevated in OA chondrocytes upon IL-1ß stimulation. However, its biological function in OA is still not fully understood. Methods: The circRNAs-miRNA-mRNA network was predicted by bioinformatics analysis. An in vitro OA chondrocytes model was established by IL-1ß stimulation. The expression of circSEC24A, miR-107-5p, CASP3, apoptosis-related molecules and extracellular matrix (ECM) components were detected by Western blot and qRT-PCR. MTT assay and Annexin V/PI staining were employed to monitor cell viability and apoptosis, respectively. The interaction between circSEC24A and miR-107-5p, as well as the binding between miR-107-5p and CASP3 3' UTR were detected by luciferase reporter and RIP assays. Cytokine secretion was monitored by ELISA assay. The role of circSEC24A was also explored in anterior cruciate ligament transection (ACLT) rat models. Results: CircSEC24A and CASP3 were increased, but miR-107-5p was decreased in rat OA cartilage tissues and OA chondrocytes. CircSEC24A acted as a sponge of miR-107-5p. Knockdown of circSEC24A promoted chondrocyte proliferation, but suppressed chondrocyte apoptosis, ECM degradation and inflammation via sponging miR-107-5p. CASP3 was identified as a miR-107-5p target gene. MiR-107-5p mimics protected against OA progression via targeting CASP3. Silencing of circSEC24A alleviated OA progression in ACLT model. Conclusion: CircSEC24A promotes OA progression through miR-107-5p/CASP3 axis.

Morphological and life-history trait plasticity of two Daphnia species induced by fish kairomones.

Daphnia can avoid predation by sensing fish kairomones and producing inducible defenses by altering the phenotype. In this study, the results showed that the morphological and life-history strategies of two Daphnia species (Daphnia pulex and Daphnia sinensis) exposed to Aristichthys nobilis kairomones. In the presence of fish kairomones, the two Daphnia species exhibited significantly smaller body length at maturity, smaller body length of offspring at the 10th instar, and longer relative tail spine of offspring. Nevertheless, other morphological and life-history traits of the two Daphnia species differed. D. pulex showed a significantly longer relative tail spine length and earlier age at maturity after exposure to fish kairomones. The total offspring number of D. sinensis exposed to fish kairomones was significantly higher than that of the control group, whereas that of D. pulex was significantly lower. These results suggest that the two Daphnia species have different inducible defense strategies (e.g., morphological and life-history traits) during prolonged exposure to A. nobilis kairomones, and their offspring also develop morphological defenses to avoid predation. It will provide reference for further exploring the adaptive evolution of Daphnia morphology and life-history traits in the presence of planktivorous fish.

Full-angle single-shot quantitative phase imaging based on Kramers-Kronig relations.

As a non-interference and non-iterative method, annular-illumination quantitative phase imaging based on Kramers-Kronig relations (AIKK) can realize phase measurement with full-angle resolution enhancement under multiple exposures. In order to completely record the object spectrum with a single shot, we proposed a colorful complementary illumination method in the recording process. The angle of this illumination mode is not symmetrical with each other, so the spectrum between the three channels can complement each other to avoid spectrum loss caused by spectrum conjugation. Meanwhile, the three spectral segments of full-angle information spectrum respectively carried by three wavelengths can be recorded. Additionally, the numerical filter is applied to correct the overlapped spectrum in the reconstruction process. Simulation and experimental results show that this method can achieve high spatiotemporal resolution quantitative phase measurement.

Recent advances in C(sp3)-N bond formation via metallaphoto-redox catalysis.

The C(sp3)-N bond is ubiquitous in natural products, pharmaceuticals, biologically active molecules and functional materials. Consequently, the development of practical and efficient methods for C(sp3)-N bond formation has attracted more and more attention. Compared to the conventional ionic pathway-based thermal methods, photochemical processes that proceed through radical mechanisms by merging photoredox and transition-metal catalyses have emerged as powerful and alternative tools for C(sp3)-N bond formation. In this review, recent advances in the burgeoning field of C(sp3)-N bond formation via metallaphotoredox catalysis have been highlighted. The contents of this review are categorized according to the transition metals used (copper, nickel, cobalt, palladium, and iron) together with photocatalysis. Emphasis is placed on methodology achievements and mechanistic insight, aiming to inspire chemists to invent more efficient radical-involved C(sp3)-N bond-forming reactions.

Photoinduced Copper-Catalyzed Asymmetric Three-Component Radical 1,2-Azidooxygenation of 1,3-Dienes.

Radical-involved multicomponent difunctionalization of 1,3-dienes has recently emerged as a promising strategy for rapid synthesis of valuable allylic compounds in one-pot operation. However, the expansion of radical scope and enantiocontrol remain two major challenges. Herein, we describe an unprecedented photoinduced copper-catalyzed highly enantioselective three-component radical 1,2-azidooxygenation of 1,3-dienes with readily available azidobenziodazolone reagent and carboxylic acids. This mild protocol exhibits a broad substrate scope, high functional group tolerance, and exceptional control over chemo-, regio- and enantioselectivity, providing practical access to diverse valuable azidated chiral allylic esters. Mechanistic studies imply that the chiral copper complex is implicated as a bifunctional catalyst in both the photoredox catalyzed azidyl radical generation and enantioselective radical C-O cross-coupling.

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.