Blood pressure states transitions among bus drivers: the application of multi-state Markov model.

PURPOSE: To explore the transition patterns among different levels of blood pressure for bus drivers. METHODS: A follow-up study was conducted to investigate the transition probabilities of three blood pressure states, the sojourn time in each state, the survival probabilities, and the risk factors affecting hypertension progression. Based on 5618 blood pressure measurements of 1883 Chinese bus drivers from 2017 to 2021, a multi-state Markov model was established. RESULTS: The mean age of participants was 41.43 ± 6.71 years, and 82.37% were male. The probabilities from normal state to hypertension within 1-year follow-up were 4.60% (male) and 2.13% (female); however, the corresponding changes over a 3-year follow-up had a significant increase to 20.27% (male) and 7.98% (female). The sojourn time of normal state and elevated state was 1.82 ± 0.09 years and 1.98 ± 0.08 years, respectively. The male participants, the increasing age, BMI, CHOL and TG were risk factors for progression from normal state to elevated state, and/or from elevated state to hypertensive state (HR: 1.04-2.53). CONCLUSIONS: Early preventive measures on the progression of hypertension should be taken for Chinese bus drivers, and at least one physical examination per year is strongly recommended. The government and bus companies should pay more attention to these kinds of bus drivers with high probabilities of hypertension: male, older than 40 years, with the higher values of BMI and blood lipid. The derived findings of sojourn time, transition probabilities and survival probabilities can provide reference for health professionals to make targeted intervention to reduce bus drivers' hypertension occurrence.

MiR-122-5p knockdown protects against APAP-mediated liver injury through up-regulating NDRG3.

MiR-122-5p serves as a novel biomarker for drug-induced liver injury (DILI), but its function in DILI remains unclear. The present study, therefore, explored the function and potential mechanism of miR-122-5p in DILI. Sprague-Dawley (SD) rats were treated with miR-122-5p antagomir, and then DILI was induced in the rats by acetaminophen (APAP). To determine the effect of miR-122-5p on DILI in vivo, liver injury was examined by HE staining and TUNEL assays, and the levels of serum ALT and AST were determined using an automated clinical chemistry analyzer. To further reveal the mechanism of miR-122-5p in DILI, THLE-2 (normal liver cell line) cells were transfected with miR-122-5p mimic and inhibitor, NDRG3, and siNDRG3, and then injured by APAP. The relationship between miR-122-5p and NDRG3 was determined by TargetScan, luciferase reporter assay, and Western blot. The viability and apoptosis of THLE-2 cells were detected by CCK-8 and flow cytometry, respectively. The levels of mRNA and protein in vivo and in vitro were measured by qRT-PCR and Western blot, respectively. APAP induced liver injury and increased the levels of ALT, AST, and miR-122-5p in DILI rats. However, these effects of APAP were attenuated by miR-122-5p antagomir. MiR-122-5p negatively regulated NDRG3 expression. APAP decreased cell viability, apoptosis resistance, and Bcl-w and Bcl-2 levels whereas increased Bax level in THLE-2 cells. However, these effects of APAP on THLE-2 cells were promoted by miR-122-5p up-regulation but inhibited by miR-122-5p knockdown. MiR-122-5p knockdown protects against APAP-mediated liver injury through up-regulating NDRG3.

Y-reconstruction could be better for ACL reconstruction in knee hyperextension versus double-bundle double-tunnel technique: a retrospective comparative study of 56 patients.

PURPOSE: To compare the clinical outcomes of double-bundle (DB) single-tibial tunnel technique and double-tunnel technique for ACL reconstruction in patients with knee hyperextension. METHODS: Defined as having constitutional hyperextension of greater than 10°, 56 patients with knee hyperextension who underwent ACL reconstruction were included in this study. To exclude concomitant lesions, preoperative magnetic resonance imaging (MRI) was performed in all knees. 24 patients (Group A) were treated with the anatomic DB/single-tibial tunnel ACL reconstruction and 32 patients (Group B) were treated with DB/double-tibial tunnel ACL reconstruction, all the included patients had knee hyperextension. Clinical results were evaluated by the extension angle, ROM, IKDC 2000 subjective score, rotational stability, pivot-shift test and anterior-posterior translation test before the operation and at the end of follow-up. MRI scan of the knee positioned in full extension was performed after 6 months post-operation. Location of tibial tunnels and graft signal intensity were assessed according to the MRI. RESULTS: Postoperative extension deficit was detected in Group B, ROM of the injured knee in Group A was from extension angle 8.91 ± 3.16° to flexion angle 115.58 ± 10.53°. ROM of the injured knee in Group B was from extension angle - 2.13 ± 5.88° to flexion angle 119.25 ± 12.63°. Flexion angles of two groups did not show any significant difference (p = 0.24), while extension angles were quite different (p < 0.0001). Group A was slightly higher than Group B in IKDC subjective scores, but without significant difference (Group A 45.1 ± 6.5, Group B 42.4 ± 4.8, p = 0.09). There was no significant difference between two groups in pivot-shift test. Post-operational MRI showed more anterior located tibial tunnel and higher graft signal intensity in Group B when compared with Group A. One patient in the Group B had ligament retear, and required revision surgery. CONCLUSION: DB/single-tibial tunnel technique restored the knee stability and overcame the shortcomings (such as knee extension deficit and graft impingement) of DB/double tibial tunnel, which might be more suitable for ACL reconstruction in knees with hyperextension. LEVEL OF EVIDENCE: Level II to III.

[Establishment of near-infrared on-line monitoring model and determination of endpoint in frying process of Hordei Fructus Germinatus].

Hordei Fructus Germinatus has been always used by "stir-frying" as a traditional medicine and the endpoint judgment of "fragrant" and "yellow" has been the focus and difficulty in frying process research. In this study, a quantitative calibration model between index components and NIRS was established in order to rapidly detect the contents of reducing sugar, total amino acids, total flavonoids, A420 and moisture; besides, an endpoint judgment method of frying process was put forward based on the "component change rate". Near-infrared spectra of samples with different frying time were collected, and a quantitative analysis model based on near-infrared spectroscopy was established to optimize the parameters such as spectral pretreatment and modeling band. HCA and PLS-DA were used to study the relationship between component and "stir-frying", and the endpoint of frying process was determined. The established calibration model had a good performance that the correlation coefficients between the predicted results and the actual measured values were both more than 0.9, with predicted relative deviations less than 10%. Hordei Fructus Germinatus with different frying time was divided into 4 categories by HCA analysis. PLS-DA analysis showed that total reducing sugar had a significant contribution to distinguishing the Hordei Fructus Germinatus of different frying time. When the change rate of total reducing sugar was 80%, it could judge that the endpoint of frying had been obtained. Results showed that NIRS could not only quickly and accurately determine the contents of reducing sugar, total amino acid, total flavonoid, A420 and moisture in Hordei Fructus Germinatus samples of different frying time, but also judge the endpoint of frying in the process. This study provided a new method for the evaluation and endpoint judgment of "stir-frying" process in research of traditional Chinese medicine.

Distally Based Saphenous Nerve-Greater Saphenous Venofasciocutaneous Flap for Reconstruction of Soft Tissue Defects in Distal Lower Leg.

BACKGROUND: To this day, the reconstruction of soft tissue defects in the distal lower leg is still a challenge for reconstructive surgeons. At present, free flap, musculocutaneous flap, anterior and posterior tibial artery flap, cross-leg flap, and distally based neurocutaneous flap are the main surgical alternatives. However, these methods are not always satisfactory. This article describes our experience with the distally based saphenous nerve-greater saphenous venofasciocutaneous flap method to reconstruct soft tissue defects in the distal lower leg. METHODS: A total of 27 patients (17 men and 10 women) underwent reconstructive surgery in our department from October 2009 to July 2012. Their mean age was 44.8 years (range, 16-62 years). The mean follow-up period was 18 months (range, 12-30 months). The only etiology was acute traumatic injury. Defects were located in the distal tibia around the ankle joint and the foot. The fasciocutaneous pedicle was 3 to 4 cm wide and the anatomical structures consisted of the superficial and deep fascia, the saphenous nerve, the greater saphenous vein, and their accompanying vessels together with an islet of subcutaneous cellular tissue and skin. The pivot point was approximately 5 cm proximal to the tip of medial malleolus. The size of skin islands ranged from 7 × 5 to 14 × 10 cm. RESULTS: All flaps survived without any partial necrosis and no arterial crisis occurred postoperatively in any case. Severe venous congestion occurred only in 1 case; however, the flap got better gradually and survived eventually after limb raise and interrupted bloodletting. All donor sites were 1-stage closed with split-skin grafting. CONCLUSIONS: The reverse saphenous neurocutaneous island flap is a reliable and efficient method to reconstruct soft tissue defects in the distal lower leg. Both this flap and the classic sural flap provide valuable alternative coverage of such defects.

[Effects of combined administration of Paeoniae Radix Alba on local pharmacokinetics of six aconite alkaloids by skin microdialysis in vivo].

The Aconiti Radix Cocta gel and Aconiti Radix Cocta combined with Paeoniae Radix Alba gel were administered to mice. Physiological saline was taken as perfusate. The perfusion rate was 2 µL•min⁻¹ and the microdialysis samples were collected every 0.5 h intervals for eight times. The six aconitine alkaloids concentration in perfusate were determined by HPLC-MS/MS. The concentration-time curves were plotted, and the pharmacokinetic parameters were calculated and analyzed by SPSS. The effects of Paeoniae Radix Alba on transdermal permeation role of six aconitine alkaloids in herb couple of Paeoniae Radix Alba-Aconiti Radix Cocta were investigated. According to the results, Tmax of the three mono-ester aconitum alkaloids of Aconiti Radix Cocta combined with Paeoniae Radix Alba groups were shortened, meanwhile, Cmax and AUC of benzoylaconine and benzoylhypaconine were increased. However, AUC of the three diester-type alkaloids were reduced, with Tmax of hypaconitine prolonged and Cmax lowered. The study suggested that the combined administration of Aconiti Radix Cocta and Paeoniae Radix Alba promoted the transdermal permeation of mono-ester aconitum alkaloids, and inhibited the absorption of parts of diester-type alkaloids. This study proved the decreasing toxicity and increasing efficacy of the combination of Aconiti Radix Cocta and Paeoniae Radix Alba on the transdermal permeation, and provided a reference for studies on the prescription combination regularity and relevant practices.

[Dynamic change rules of active ingredients and inactive ingredients from Fructus Hordei Germinatus with frying process].

It is not scientific to explain that fried Fructus Hordei Germinatus is more effective than row Fructus Hordei Germinatus in resolving food stagnation from the aspects of amylase, tricine and other "active ingredients". In the present experiment, the contents of active ingredients including quercetin, tricine, kaempferol, catechin, ferulic acid and inactive ingredients including 5-hydroxymethyl furfural, acrylamide in frying process were determined by HPLC. The dynamic change rules of active ingredient and inactive ingredients in the frying process were investigated by HCA, PCA and PLS-DA analysis. The results showed that the Fructus Hordei Germinatus samples with different frying temperatures were classified into 4 groups by HCA and PCA analysis. PLS-DA analysis showed that frying temperature mainly impacted the contents of inactive ingredients including 5-hydroxymethyl furfural and acrylamide, with less effects on the contents of active ingredients. Simultaneously, with the increase of time in frying process, the content of 5-hydroxymethyl furfural was significantly increased from 2 min and became stable at 16 min, while the content of acrylamide was increased continuously from 18 min. Based on the variation of the contents of various ingredients, samples at different frying time were classified into 5 groups. The results showed that the content changes of "inactive ingredients" were closely related to the duration and degree of frying process, and the dynamic change rules of "inactive ingredients" can provide scientific basis for evaluating the frying process and elucidating the efficacy mechanism of Fructus Hordei Germinatus.

Experimental study of PLLA/INH slow release implant fabricated by three dimensional printing technique and drug release characteristics in vitro.

BACKGROUND: Local slow release implant provided long term and stable drug release in the lesion. The objective of this study was to fabricate biodegradable slow release INH/PLLA tablet via 3 dimensional printing technique (3DP) and to compare the drug release characteristics of three different structured tablets in vitro. METHODS: Three different drug delivery systems (columnar-shaped tablet (CST), doughnut-shaped tablet (DST) and multilayer doughnut-shaped tablet (MDST)) were manufactured by the three dimensional printing machine and isoniazid was loaded into the implant. Dynamic soaking method was used to study the drug release characteristics of the three implants. MTT cytotoxicity test and direct contact test were utilized to study the biocompatibility of the implant. The microstructures of the implants' surfaces were observed with electron microscope. RESULTS: The PLLA powder in the tablet could be excellently combined through 3DP without disintegration. Electron microscope observations showed that INH distributed evenly on the surface of the tablet in a "nest-shaped" way, while the surface of the barrier layer in the multilayer doughnut shaped tablet was compact and did not contain INH. The concentration of INH in all of the three tablets were still higher than the effective bacteriostasis concentration (Isoniazid: 0.025 ~ 0.05 µg/ml) after 30 day's release in vitro. All of the tablets showed initial burst release of the INH in the early period. Drug concentration of MDST became stable and had little fluctuation starting from the 6th day of the release. Drug concentration of DST and CST decreased gradually and the rate of decrease in concentration was faster in DST than CST. MTT cytotoxicity test and direct contact test indicated that the INH-PLLA tablet had low cytotoxicity and favorable biocompatibility. CONCLUSIONS: Three dimensional printing technique was a reliable technique to fabricate complicated implants. Drug release pattern in MDST was the most stable among the three implants. It was an ideal drug delivery system for the antibiotics. Biocompatibility tests demonstrated that the INH-PLLA implant did not have cytotoxicity. The multilayer donut-shaped tablet provided a new constant slow release method after an initial burst for the topical application of the antibiotic.

Chimeric antigen receptor therapy meets mRNA technology.

Genetically engineered immune cells expressing chimeric antigen receptors (CARs) have emerged as a new game changer in cancer immunotherapy. The utility of CAR T cell therapy against hematological malignancies has been validated in clinical practice. Other CAR immune cells are currently under investigation to improve the potency of CAR therapy in solid tumors. As a new class of therapeutic modalities, mRNA-based therapeutics hold enormous potential beyond COVID-19 mRNA vaccines. Arming immune cells with mRNA-encoded CARs represents a new frontier in cancer and beyond, enabling in vivo generation of CAR cells without causing transgene integration. In this review, we summarize recent advances in mRNA-based CAR immunotherapies and highlight their opportunities and challenges for the development of a new generation of living drugs.

ACOT7 promotes retinoblastoma resistance to vincristine by regulating fatty acid metabolism reprogramming.

The rate of vincristine (VCR) resistance in the treatment of retinoblastoma (RB) is relatively high, and the exact role and mechanism of autophagy and fatty acid (FA) metabolism in RB are still unknown. The aim of this study was to elucidate the molecular mechanism by which acyl-CoA thioesterase 7 (ACOT7) regulates FA metabolism and autophagy, which may lead to potential therapeutic strategies for RB. In the present study, the relationship between FA metabolism and cellular drug sensitivity was evaluated through ACOT7 overexpression or inhibition tests in RB-resistant cells. The lipase inhibitor orlistat and the autophagy inhibitor CQ were used to determine the effects of ACOT7 on FA metabolism, autophagy, and cellular drug sensitivity, as well as the therapeutic value of ACOT7 targeting. The results showed that ACOT7 was upregulated in VCR-resistant RB cells, significantly enhancing cell resistance and indicating that ACOT7 may serve as a biomarker for VCR resistance in RB cells. Knockdown of ACOT7 inhibited FA metabolism and reduced cell viability in VCR-resistant RB cells. The effect of ACOT7 overexpression was opposite to that of ACOT7 knockdown, and ACOT7 overexpression promoted autophagy in VCR-resistant RB cells. After treatment with orlistat or CQ, FA metabolism in VCR-resistant RB cells decreased, cell viability and autophagy were inhibited, EMT was inhibited, and the sensitivity of RB cells to VCR was increased. In conclusion, ACOT7 knockdown can mediate FA metabolism to inhibit autophagy and the migration of RB cells, thereby improving the sensitivity of RB cells to VCR.

Discussion on treatment courses of brucellosis with spondylitis - a report of two cases.

Human Brucellosis is a zoonotic contagious disease caused by Brucella infection and is common throughout the world, which can travel through the bloodstream to various organs. Brucellar spondylitis(BS) is the foremost cause of brucellosis's debilitating and disabling complications. We report two sisters with brucellosis complicated by lumbar spondylodiscitis accompanied by cold abscess formation. The diagnosis was based on their symptoms, epidemiological characteristics, laboratory and magnetic resonance imaging(MRI) results. Our therapeutic strategy in these two cases indicate that drug combination and prolongation of the use of antibiotics is a therapeutic strategy worthy of popularizing to reach a greater clearance rate of the infection.

A PEG-lipid-free COVID-19 mRNA vaccine triggers robust immune responses in mice.

COVID-19 mRNA vaccines represent a completely new category of vaccines and play a crucial role in controlling the COVID-19 pandemic. In this study, we have developed a PEG-lipid-free two-component mRNA vaccine (PFTCmvac) by formulating mRNA encoding the receptor binding domain (RBD) of SARS-CoV-2 into lipid-like nanoassemblies. Without using polyethylene glycol (PEG)-lipids, the self-assembled PFTCmvac forms thermostable nanoassemblies and exhibits a dose-dependent cellular uptake and membrane disruption, eventually leading to high-level protein expression in both mammalian cells and mice. Vaccination with PFTCmvac elicits strong humoral and cellular responses in mice, without evidence of significant adverse reactions. In addition, the vaccine platform does not trigger complement activation in human serum, even at a high serum concentration. Collectively, the PEG-lipid-free two-component nanoassemblies provide an alternative delivery technology for COVID-19 mRNA vaccines and opportunities for the rapid production of new mRNA vaccines against emerging infectious diseases.

Induction of Resistance of Antagonistic Bacterium Burkholderia contaminans to Postharvest Botrytis cinerea in Rosa vinifera.

In order to study the problem that grapes are vulnerable to microbial infection and decay during storage, a method based on antagonistic Burkholderia contaminans against postharvest Botrytis cinerea of Rosa vinifera was proposed in this paper. The method tested the resistance induction mechanism of Botrytis cinerea after harvest and determined the fruit decay rate treated by antagonistic Burkholderia contaminans. The results showed that the antagonistic bacterium B-1 had bacteriostatic effect on many common pathogens of fruits and vegetables to a certain extent, and the bacteriostatic range was wide. Among them, the inhibition rate of Fusarium moniliforme was 75.5% and that of Botrytis cinerea was 51.2%. After testing, it can be found that antagonistic bacteria have an inhibitory effect on pathogenic fungi and have an effect on phenylpropane metabolic pathway, reactive oxygen species metabolic pathway, and the activities of other resistance-related enzymes. Through comparison, it can be found that the antagonistic Burkholderia contaminans has a strong antibacterial mechanism against Botrytis cinerea of rose grape after harvest. The fruit treated with antagonistic B Burkholderia B-1 has significantly reduced the decay rate and increased the activity of antibacterial active protein.

Altered fecal microbial and metabolic profile reveals potential mechanisms underlying iron deficiency anemia in pregnant women in China.

The gut microbiome and its metabolism may provide crucial insight into the cause of iron deficiency anemia (IDA) in pregnant women. This study aimed to investigate the effect of the gut microbiome and its related metabolites on pregnant women with iron deficiency (ID) and IDA. Maternal cubital venous blood and stool samples were collected from healthy control pregnant women (HC, non-anemic, n=10), pregnant women with ID non-anemia (ID, n=10), and IDA (n=10). All groups were subjected to fecal metagenomics and metabolomics. The composition and function of the gut microbiome were then compared in pregnant women with ID and IDA with HC after excluding the possibility of inflammation and insufficient iron absorption capacity. Whole-genome shotgun libraries were prepared by quantifying metagenomic DNA samples with Quant-iT PicoGreen dsDNA Assay. The levels of 41 microbial species, including 21 Streptococci and ten metabolites (catechol), which could serve as siderophores, were increased. In contrast, 3 Bacteroides and six metabolites were decreased in pregnant women with IDA (p<0.05). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the bio-pathways, including biosynthesis of siderophore group non-ribosomal peptides (p<0.01), ABC transporters (p<0.05) and membrane transport of the gut microbiota (p<0.01) in IDA patients were expressed differently compared with HC. Correlation analysis also indicates that these increased bacteria formed strong co-occurring relationships with metabolites in the occurrence and development of IDA in pregnant women. The current study identified that streptococci and catechol (fecal metabolite) were significantly increased in pregnant women with IDA. Therefore, adjusting the intestinal homeostasis using long-term living and eating habits on oral Streptococcus in pregnant women with IDA before iron supplementation may be more conducive to iron supplementation, thus providing novel therapies for IDA.

A Case of Secondary Pulmonary Syphilis - The Utility of mNGS in Bronchoalveolar Lavage Fluid: A Case Report.

Syphilis is a sexually transmitted infectious disease caused by Treponema pallidum. Here, we report a rare case of secondary pulmonary syphilis, which was diagnosed by metagenomic next-generation sequencing (mNGS) in bronchoalveolar lavage fluid (BALF). Direct pulmonary involvement by T. pallidum was suggested by a positive mNGS result in BALF. One month after treatment with benzathine penicillin G (2.4 million units, three doses), a repeated CT scan showed the radiological resolution. To our knowledge, this is the first reported case of secondary pulmonary syphilis diagnosed by mNGS in BALF.

FABP4 knockdown suppresses inflammation, apoptosis and extracellular matrix degradation in IL-1ß-induced chondrocytes by activating PPARγ to regulate the NF-κB signaling pathway.

Osteoarthritis (OA) is a common degenerative disease that can lead to severe joint pain and loss of function, seriously threatening the health and normal life of patients. At present, the pathogenesis of OA remains to be clarified. Recent studies have shown that fatty acid­binding protein 4 (FABP4) is increased in the plasma and synovial fluid of patients with OA. However, the effect of FABP4 on OA is unclear. The present study established IL­1ß­induced ATDC5 cells with FABP4 knockdown. Next, cell viability was detected with Cell Counting Kit­8 assay. The content of inflammatory factors, prostaglandin E2 and glycosaminoglycan (GAG) was detected via ELISA. The levels of reactive oxygen species (ROS) and superoxide dismutase (SOD) in cells were detected by using ROS and SOD kits, respectively. TUNEL staining was used to detect the apoptosis level. Western blotting was used to detect the expression levels of proteins. The results revealed that FABP4 was upregulated in IL­1ß­induced ATDC5 cells. Knockdown of FABP4 increased cell viability, reduced inflammatory damage, oxidative stress and apoptosis in IL­1ß­induced ATDC5 cells. Following FABP4 knockdown, the expression of matrix metalloproteinases (MMP3, MMP9 and MMP13) of IL­1ß­induced ATDC5 cells was reduced, and the expression of GAG was promoted. FABP4 knockdown also inhibited the expression of NF­κB p65 and enhanced peroxisome proliferator­activated receptor (PPAR)γ expression. However, the presence of PPARγ inhibitor blocked the aforementioned effects of FABP4 on IL­1ß­induced ATDC5 cells. In conclusion, FABP4 knockdown suppressed the inflammation, oxidative stress, apoptosis and extracellular matrix degradation of IL­1ß­induced chondrocytes by activating PPARγ to inhibit the NF­κB signaling pathway.

Study on the occurrence law and green control of grape gray mold from the perspective of ecological balance.

With the increase of grape planting years, the base number of pathogenic seedlings and insect population is gradually rising. In addition, the introduction, breeding system and control of seedlings are not standardized and other human factors, the occurrence of Botrytis cinerea(B.cinerea) on grape is becoming more and more serious, resulting in a prominent problem of yield decline. In this paper, the occurrence of B.cinerea was monitored and its control effect was tested from the perspective of ecological balance. Finally, the biological characteristics and control of B.cinerea were studied. The spore catcher was used to catch the pathogen spores of B. cinerea, and the amount of sporangium scattering reached its peak from August to September Spore scattering is affected by meteorological factors, and the temperature has reached a very significant level, and the low temperature and high humidity conditions are conducive to the disease; The results showed that the resistance frequency of 304 B.cinereastrains to carbendazim, boscalid, pyrimethanil was higher than 50%; the volatile compounds produced by yeast (Trichosproom sp.) YE-3-2 significantly inhibited the growth of B.cinerea (inhibition rate was 62.93%, according to the occurrence regularity of B.cinerea, the accurate and effective agricultural measures had a good control effect on B.cinerea, which could improve the quality of grape fruit and provide some help for the prevention of grape gray mold.

Secreted Expression of mRNA-Encoded Truncated ACE2 Variants for SARS-CoV-2 via Lipid-Like Nanoassemblies.

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.

Biocompatibility of KLD-12 peptide hydrogel as a scaffold in tissue engineering of intervertebral discs in rabbits.

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH(2) was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/PI fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% (mean: 92.15%+/-1.17%) at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups (complete culture media) (P>0.05) at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs, and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.

Knockdown of NHP2 inhibits hepatitis B virus X protein-induced hepatocarcinogenesis via repressing TERT expression and disrupting the stability of telomerase complex.

Hepatitis B virus X protein (HBx) is highly expressed in HBV-infected hepatocellular carcinoma (HCC) and upregulates transcriptional activation of telomerase reverse transcriptase (TERT). NHP2 is a component of the telomerase complex and also increased in HCC. However, whether NHP2 could accelerate HCC caused by HBx overexpression remains unknown. This study intended to investigate the effects of NHP2 knockdown on HBx-overexpressed HCC and uncover the potential mechanism. Results showed that after HBx overexpression, the expression of TERT and NHP2 was increased. NHP2 knockdown inhibited cell proliferation, colony formation and telomerase activity, while promoting cell apoptosis in PLC/PRF5 cells with or without HBx overexpression. Moreover, the protein expression of TERT and HBx was inhibited, pro-apoptotic proteins Bax and cleaved-caspase3 expression was enhanced, whereas anti-apoptotic protein Bcl-2 level was reduced upon NHP2 silencing in PLC/PRF5 cells with or without HBx upregulation. The interaction between NHP2 and TERT was also confirmed. Treatment with shRNA-NHP2-1 inhibited tumor growth in xenograft model, and the alterations of related proteins were consisted with in vitro results. In conclusion, knockdown of NHP2 could inhibit the proliferation of hepatoma cells overexpressing HBx via inhibiting TERT expression.