A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

Differentiating Microplastics from Natural Particles in Aqueous Suspensions Using Flow Cytometry with Machine Learning.

Microplastics (MPs) in natural waters are heterogeneously mixed with other natural particles including algal cells and suspended sediments. An easy-to-use and rapid method for directly measuring and distinguishing MPs from other naturally present colloids in the environment would expedite analytical workflows. Here, we established a database of MP scattering and fluorescence properties, either alone or in mixtures with natural particles, by stain-free flow cytometry. The resulting high-dimensional data were analyzed using machine learning approaches, either unsupervised (e.g., viSNE) or supervised (e.g., random forest algorithms). We assessed our approach in identifying and quantifying model MPs of diverse sizes, morphologies, and polymer compositions in various suspensions including phototrophic microorganisms, suspended biofilms, mineral particles, and sediment. We could precisely quantify MPs in microbial phototrophs and natural sediments with high organic carbon by both machine learning models (identification accuracies over 93%), although it was not possible to distinguish between different MP sizes or polymer compositions. By testing the resulting method in environmental samples through spiking MPs into freshwater samples, we further highlight the applicability of the method to be used as a rapid screening tool for MPs. Collectively, this workflow can be easily applied to a diverse set of samples to assess the presence of MPs in a time-efficient manner.

Intra-articular Histone Deacetylase Inhibitor Microcarrier Delivery to Reduce Osteoarthritis.

The histone deacetylase inhibitor (HDACi) was a milestone in the treatment of refractory T-cell lymphoma. However, the beneficial effects of HDACi have not been appreciated in osteoarthritis (OA). Herein, we implemented a microcarrier system because of the outstanding advantages of controlled and sustained release, biodegradability, and biocompatibility. The poly(d,l-lactide-co-glycolide) (PLGA) microcapsules have a regulated and sustained release profile with a reduced initial burst release, which can improve the encapsulation efficiency of the Chidamide. The emulsion solvent evaporation strategy was used to encapsulate Chidamide in PLGA microcapsules. The encapsulation of Chidamide was established by UV-vis spectra and scanning electron microscopy. Additionally, the inhibition of Tnnt3 and immune stimulation by Chidamide helped to inhibit cartilage destruction and prevent articular cartilage degeneration. Based on the results, the Chidamide in PLGA microcapsules provides a transformative therapeutic strategy for the treatment of osteoarthritis patients to relieve symptoms and protect against cartilage degeneration.

A new nanobody-enzyme fusion protein-linked immunoassay for detecting antibodies against influenza A virus in different species.

Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species.

A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics.

Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention. KEY POINTS: • The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes. • A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity. • The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.

FoRSR1 Is Important for Conidiation, Fusaric Acid Production, and Pathogenicity in Fusarium oxysporum f. sp. ginseng.

The root rot disease caused by Fusarium oxysporum f. sp. ginseng is one of the most destructive diseases of ginseng, an economically important herb. However, little is known about the pathogen's toxin biosynthesis or the molecular mechanisms regulating infection of ginseng. In this study we identified and functionally characterized the FoRSR1 gene that encodes a Ras-related (RSR) small GTPase homologous to yeast Rsr1 in F. oxysporum f. sp. ginseng. Disruption of FoRSR1 resulted in a significant reduction in mycelial dry weight in liquid cultures, although vegetative growth rate was not affected on culture plates. Notably, the Forsr1 mutant exhibited blunted and swollen hyphae with multi-nucleated compartments. It produced fewer and morphologically abnormal conidia and was defective in chlamydospore formation. In infection assays with ginseng roots, the Forsr1 mutant was significantly less virulent and caused only limited necrosis at the wounding sites. Deletion of FoRSR1 also affected pigmentation, autophagy, and production of fusaric acid. Furthermore, the expression of many candidate genes involved in secondary metabolism was significantly downregulated in the mutant, suggesting that FoRSR1 is also important for secondary metabolism. Overall, our results indicated that FoRSR1 plays important roles in conidiation, vacuolar morphology, secondary metabolism, and pathogenesis in F. oxysporum f. sp. ginseng.

CT and MRI measurements of tibial tubercle lateralization in patients with patellar dislocation were not equivalent but could be interchangeable.

PURPOSE: To compare the values and the relationship of tibial tubercle lateralization measurements between computerized tomography (CT) and magnetic resonance imaging (MRI). METHODS: Sixty patients with patellar dislocation who underwent both CT and MRI of the same knee joint from November 2021 to February 2022 were included in our study. The intraclass correlation coefficient (ICC) and Bland-Altman analysis were performed to evaluate the reliability of tibial tubercle-trochlear groove (TT-TG), tibial tubercle-Roman arch (TT-RA), and tibial tubercle-posterior cruciate ligament (TT-PCL) distance measurements. The values of CT and MRI measurements using the same bony landmarks were compared for the difference. Pearson correlation analysis and linear regression analysis were performed to assess the correlation between CT and MRI measurements. Finally, the estimated values obtained from the regression equation were compared with the actual values obtained from the radiological measurement to evaluate the accuracy of the equations. RESULTS: A total of 60 patients with patellar dislocation who underwent both CT and MRI of the same knee joint were included in this study. The included measurements showed excellent agreement with ICCs > 0.9. TT-TG distance measured on CT (19.5 ± 5.1 mm) had a mean of 7.1 mm higher than that on MRI (12.4 ± 4.7 mm) (P < 0.001). The mean value of TT-RA distance was 22.5 ± 3.7 mm on CT and 16.7 ± 4.9 mm on MRI (P < 0.001), showing a mean difference of 5.8 mm. The values of TT-TG distance measured by CT and MRI were significantly correlated (R = 0.5, P < 0.001). The values of TT-RA distance between these two modalities showed a better correlation than that of TT-TG distance (R = 0.6, P < 0.001). The interchange values of TT-TG distance and TT-RA distance between CT and MRI can be obtained using regression equations (TT-TG distance: y = 0.6x + 12.3; TT-RA distance: y = 0.5x + 14.4). CONCLUSION: The values of tibial tubercle lateralization measured by MRI may be underestimated compared with those measured by CT. Although the values measured on CT and MRI are not equivalent, the value in the other modality can be estimated. Therefore, an additional CT scan for tibial tubercle lateralization evaluation may not be necessary. LEVEL OF EVIDENCE: Level II.

An MMIC LNA for Millimeter-Wave Radar and 5G Applications with GaN-on-SiC Technology.

This paper presents a monolithic microwave integrated circuit (MMIC) low noise amplifier (LNA) that is compatible with n257 (26.5-29.5 GHz) and n258 (24.25-27.5 GHz) frequency bands for fifth-generation mobile communications system (5G) and millimeter-wave radar. The total circuit size of the LNA is 2.5 × 1.5 mm2. To guarantee a trade-off between noise figure (NF) and small signal gain, the transmission lines are connected to the source of gallium nitride (GaN)-on-SiC high electron mobility transistors (HEMT) by analyzing the nonlinear small signal equivalent circuit. A series of stability enhancement measures including source degeneration, an RC series network, and RF choke are put forward to enhance the stability of designed LNA. The designed GaN-based MMIC LNA adopts hybrid-matching networks (MNs) with co-design strategy to realize low NF and broadband characteristics across 5G n257 and n258 frequency band. Due to the different priorities of these hybrid-MNs, distinguished design strategies are employed to benefit small signal gain, input-output return loss, and NF performance. In order to meet the testing conditions of MMIC, an impeccable system for measuring small has been built to ensure the accuracy of the measured results. According to the measured results for small signal, the three-stage MMIC LNA has a linear gain of 18.2-20.3 dB and an NF of 2.5-3.1 dB with an input-output return loss better than 10 dB in the whole n257 and n258 frequency bands.

Customized Trajectory Optimization and Compliant Tracking Control for Passive Upper Limb Rehabilitation.

Passive rehabilitation training in the early poststroke period can promote the reshaping of the nervous system. The trajectory should integrate the physicians' experience and the patient's characteristics. And the training should have high accuracy on the premise of safety. Therefore, trajectory customization, optimization, and tracking control algorithms are conducted based on a new upper limb rehabilitation robot. First, joint friction and initial load were identified and compensated. The admittance algorithm was used to realize the trajectory customization. Second, the improved butterfly optimization algorithm (BOA) was used to optimize the nonuniform rational B-spline fitting curve (NURBS). Then, a variable gain control strategy is designed, which enables the robot to track the trajectory well with small human-robot interaction (HRI) forces and to comply with a large HRI force to ensure safety. Regarding the return motion, an error subdivision method is designed to slow the return movement. The results showed that the customization force is less than 6 N. The trajectory tracking error is within 12 mm without a large HRI force. The control gain starts to decrease in 0.5 s periods while there is a large HRI force, thereby improving safety. With the decrease in HRI force, the real position can return to the desired trajectory slowly, which makes the patient feel comfortable.

Robust Sparse Bayesian Two-Dimensional Direction-of-Arrival Estimation with Gain-Phase Errors.

To reduce the influence of gain-phase errors and improve the performance of direction-of-arrival (DOA) estimation, a robust sparse Bayesian two-dimensional (2D) DOA estimation method with gain-phase errors is proposed for L-shaped sensor arrays. The proposed method introduces an auxiliary angle to transform the 2D DOA estimation problem into two 1D angle estimation problems. A sparse representation model with gain-phase errors is constructed using the diagonal element vector of the cross-correlation covariance matrix of two submatrices of the L-shaped sensor array. The expectation maximization algorithm derives unknown parameter expression, which is used for iterative operations to obtain off-grid and signal precision. Using these parameters, a new spatial spectral function is constructed to estimate the auxiliary angle. The obtained auxiliary angle is substituted into a sparse representation model with gain and phase errors, and then the sparse Bayesian learning method is used to estimate the elevation angle of the incident signal. Finally, according to the relationship of the three angles, the azimuth angle can be estimated. The simulation results show that the proposed method can effectively realize the automatic matching of the azimuth and elevation angles of the incident signal, and improves the accuracy of DOA estimation and angular resolution.

Kaempferol ameliorates in-vitro and in-vivo postovulatory oocyte ageing in mice.

RESEARCH QUESTION: Does kaempferol alleviate postovulatory oocyte ageing, thereby maintaining their early embryonic development capacity? DESIGN: The effects of kaempferol on postovulatory ageing were investigated in vitro and in vivo by short-term kaempferol administration (mature oocytes were cultured in a kaempferol-containing medium for 12 h; mice were intragastrically administered with the appropriate amount of kaempferol for 21 days). Spindle morphology and chromosome alignment, levels of oxidative stress and the gap junction were assessed by immunofluorescence. Fertilization ability and early embryonic development ability of each oocyte group was detected by IVF. Fertilization of the ageing oocyte model was used to explore whether kaempferol could improve adverse pregnancy outcome. RNA-sequencing and quantitative polymerase chain reaction were used to identify the cellular pathways through which kaempferol relieves postovulatory oocyte ageing in vivo. RESULTS: Kaempferol administration altered various processes in the ageing oocytes, including oxidative stress, the peroxisome, TNF signalling, cAMP signalling and the gap junction pathway. Expression of several important genes, such as Sirt1, Mapk1, Ampk and Foxo3, was regulated. Moreover, kaempferol ameliorated adverse pregnancy outcomes of fertilized ageing oocytes. IVF results indicate that kaempferol could partially counteract the effects of oocyte ageing on fertilization capacity (pronucleus: kaempferol, 69.08 ± 2.37% versus aged, 38.95 ± 3.58%) and early embryonic development (blastocyst: kaempferol, 50.02 ± 3.34% versus aged, 30.83 ± 5.46%). CONCLUSIONS: Our results indicate that kaempferol may be a potent natural antioxidant, have implications for animal husbandry and may help improve the success rate of IVF and ICSI. Further clinical trials are needed.

Photoaging of Baby Bottle-Derived Polyethersulfone and Polyphenylsulfone Microplastics and the Resulting Bisphenol S Release.

This study evaluated the release of bisphenol S (BPS) from polyethersulfone (PES) and polyphenylsulfone microplastics (MPs) derived from baby bottles under UV irradiation. Released BPS fluctuates over time because it undergoes photolysis under UV254 irradiation. Under UV365 irradiation, the highest released concentration at 50 °C was 1.7 and 3.2 times that at 35 and 25 °C, respectively, as the activation energy of the photochemical reactions responsible for MP decay was reduced at high temperatures. Low concentrations of humic acid (HA, ≤10 mg·L-1) promote BPS release because HA acts as a photosensitizer. A high concentration of HA (10∼50 mg·L-1) decreases the BPS release because HA shields MPs from light and scavenges reactive radicals that are produced via photochemical reactions. For example, under UV irradiation, hydroxyl radicals (•OH) attack results in the breakage of ether bonds and the formation of phenyl radicals (Ph•) and phenoxy radicals (Ph-O•).The•OH addition and hydrogen extractions further produce BPS from the decayed MPs. A leaching kinetics model was developed and calibrated by the experimental data. The calibrated model predicts the equilibrium level of BPS release from MPs that varies with the surface coverage density of BPS and leaching rate constants. This study provides groundwork that deepens our understanding of environmental aging and the chemical release of MPs.

Accurate Channel Estimation and Adaptive Underwater Acoustic Communications Based on Gaussian Likelihood and Constellation Aggregation.

Achieving accurate channel estimation and adaptive communications with moving transceivers is challenging due to rapid changes in the underwater acoustic channels. We achieve an accurate channel estimation of fast time-varying underwater acoustic channels by using the superimposed training scheme with a powerful channel estimation algorithm and turbo equalization, where the training sequence and the symbol sequence are linearly superimposed. To realize this, we develop a 'global' channel estimation algorithm based on Gaussian likelihood, where the channel correlation between (among) the segments is fully exploited by using the product of the Gaussian probability-density functions of the segments, thereby realizing an ideal channel estimation of each segment. Moreover, the Gaussian-likelihood-based channel estimation is embedded in turbo equalization, where the information exchange between the equalizer and the decoder is carried out in an iterative manner to achieve an accurate channel estimation of each segment. In addition, an adaptive communication algorithm based on constellation aggregation is proposed to resist the severe fast time-varying multipath interference and environmental noise, where the encoding rate is automatically determined for reliable underwater acoustic communications according to the constellation aggregation degree of equalization results. Field experiments with moving transceivers (the communication distance was approximately 5.5 km) were carried out in the Yellow Sea in 2021, and the experimental results verify the effectiveness of the two proposed algorithms.

Molecular targeting therapies for neuroblastoma: Progress and challenges.

There is an urgent need to identify novel therapies for childhood cancers. Neuroblastoma is the most common pediatric solid tumor, and accounts for ~15% of childhood cancer-related mortality. Neuroblastomas exhibit genetic, morphological and clinical heterogeneity, which limits the efficacy of existing treatment modalities. Gaining detailed knowledge of the molecular signatures and genetic variations involved in the pathogenesis of neuroblastoma is necessary to develop safer and more effective treatments for this devastating disease. Recent studies with advanced high-throughput "omics" techniques have revealed numerous genetic/genomic alterations and dysfunctional pathways that drive the onset, growth, progression, and resistance of neuroblastoma to therapy. A variety of molecular signatures are being evaluated to better understand the disease, with many of them being used as targets to develop new treatments for neuroblastoma patients. In this review, we have summarized the contemporary understanding of the molecular pathways and genetic aberrations, such as those in MYCN, BIRC5, PHOX2B, and LIN28B, involved in the pathogenesis of neuroblastoma, and provide a comprehensive overview of the molecular targeted therapies under preclinical and clinical investigations, particularly those targeting ALK signaling, MDM2, PI3K/Akt/mTOR and RAS-MAPK pathways, as well as epigenetic regulators. We also give insights on the use of combination therapies involving novel agents that target various pathways. Further, we discuss the future directions that would help identify novel targets and therapeutics and improve the currently available therapies, enhancing the treatment outcomes and survival of patients with neuroblastoma.

Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.

Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.

Perfluorooctane sulfonate affects mouse oocyte maturation in vitro by promoting oxidative stress and apoptosis induced bymitochondrial dysfunction.

Perfluorooctane sulphonate (PFOS), as a surfactant, is widely applied in the agricultural production activities and has become a potential menace to human health. The mechanism of its effect on the maturation of mammalian oocytes is unclear. This study explored the toxic effect of PFOS on mouse oocyte maturation in vitro. The results revealed that PFOS under a concentration of 600 µM could significantly reduce the polar body extrusion rate (PBE) of mouse oocytes and cause symmetrical cell division. Further experiments showed that PFOS resulted in the abnormal cytoskeleton of the oocytes, causing the abnormal spindles and misplaced chromosomes, as well as the impaired dynamics of actin. Moreover, PFOS exposure inhibited the process of oocyte meiosis, which reflected in the slower spindle migration and continuous activation of spindle assembly checkpoint (SAC), then ultimately increased the probability of aneuploidy. Most importantly, PFOS exposure reduced the quality of oocytes, specifically by disrupting the function of mitochondria, inducing cell oxidative stress, and triggering early apoptosis. Furthermore, the level of methylation of histones is additionally influenced. In summary, our findings showed that PFOS exposure interfered with the maturation of mouse oocytes through affecting cytoskeletal dynamics, meiotic progression, oocyte quality, and histone modifications.

Double-bundle anterior cruciate ligament reconstruction technique has advantages in chondroprotection and knee laxity control compared with single-bundle technique : A long-term follow-up with a minimum of 12 years.

PURPOSE: To compare the long-term clinical outcomes of single-bundle anterior cruciate ligament reconstruction (SBR) and double-bundle anterior cruciate ligament reconstruction (DBR) in patients with isolated anterior cruciate ligament (ACL) rupture, presenting no meniscus injury and no obvious preoperative cartilage degeneration. METHODS: One hundred and three patients (38.6 ± 9.5 years) with a median follow-up of 151.6 months (range, 144-189 months) completed the retrospective study (SBR group: n = 51; DBR group: n = 52). Clinical outcomes were evaluated with physical examinations, KT-2000 anterior and posterior stability measurement with the knee in 30º of flexion, International Knee Documentation Committee (IKDC) subjective score, Tegner score, Lysholm score; magnetic resonance imaging (MRI) (3.0 T) was performed, and International Cartilage Repair Society (ICRS) cartilage degeneration grades were determined. Multivariate analysis was performed to identify factors associated with cartilage degeneration. RESULTS: There were significant differences in the pre- and postoperative IKDC, Lysholm and Tegner scores between the SBR and DBR groups. The SBR group had over double the rate of positive pressure/rub patellar test results (SBR vs DBR, 43.1% vs. 19.2%, p < 0.011). The KT-2000, pivot-shift and Lachman test results were stratified and analyzed, and significant differences between the SBR and DBR groups were found (p < 0.05, respectively). The distribution of ICRS grades differed significantly between the groups at the last follow-up (p = 0.013). A multivariate analysis found that age and operation procedures were significant predictors of 0 and non-0 ICRS grades (odds ratio, 6.077 [95% CI 2.117-17.447] and 0.210 [95% CI 0.068-0.654], respectively) (p < 0.05). CONCLUSION: Both SBR and DBR achieved overall good long-term results. DBR had advantages in objective outcome measures and was superior in preventing the occurrence of cartilage degeneration. Age was identified as a preoperative risk factor for significant postoperative cartilage degeneration. LEVEL OF EVIDENCE: III. ClinicalTrials.gov: NCT03984474.

Picroside II alleviates liver injury induced by alpha-naphthylisothiocyanate through AMPK-FXR pathway.

Alpha-naphthylisothiocyanate (ANIT) is a typical hepatotoxicant that causes cholestasis, which causes toxic bile acid accumulation in the liver and leads to liver injury. Picroside II (PIC), one of the dominant effective components extracted from Picrorhiza scrophulariiflora Pennell, exhibits many pharmacological effects. However, the role of AMP-activated protein kinase (AMPK)-Farnesoid X receptor (FXR) pathway in the hepatoprotective effect of PIC against ANIT-induced cholestasis remains largely unknown. This study aimed to investigate the mechanisms of PIC on ANIT-induced cholestasis in vivo and in vitro. Our results showed that PIC protected against ANIT-induced liver injury in primary mouse hepatocytes, and decreased serum biochemical markers and lessened histological injuries in mice. ANIT inhibited FXR and its target genes of bile acid synthesis enzymes sterol-12α-hydroxylase (CYP8B1), and increase bile acid uptake transporter Na + -dependent taurocholate transporter (NTCP), efflux transporter bile salt export pump (BSEP) and bile acid metabolizing enzymes UDP-glucuronosyltransferase 1a1 (UGT1A1) expressions. PIC prevented its downregulation of FXR, NTCP, BSEP and UGT1A1, and further reduced CYP8B1 by ANIT. Furthermore, ANIT activated AMPK via ERK1/2-LKB1 pathway. PIC inhibited ERK1/2, LKB1 and AMPK phosphorylation in ANIT-induced cholestasis in vivo and in vitro. AICAR, an AMPK agonist, blocked PIC-mediated changes in FXR, CYP8B1 and BSEP expression in vitro. Meanwhile, U0126, an ERK1/2 inhibitor, further repressed ERK1/2-LKB1-AMPK pathway phosphorylation. In conclusion, PIC regulated bile acid-related transporters and enzymes to protect against ANIT-induced liver injury, which related to ERK1/2-LKB1-AMPK pathway. Thus, this study extends the understanding of the anti-cholestasis effect of PIC and provides new therapeutic targets for cholestasis treatment.

Postfusion structure of human-infecting Bourbon virus envelope glycoprotein.

Thogotoviruses are important zoonotic viruses infecting a variety of domestic animals, as well as humans. Among these viruses, Bourbon virus (BRBV) is one of the several human-infecting members, which emerged in the US in recent years and caused human deaths. Here, we report the crystal structure of the BRBV envelope glycoprotein in the postfusion conformation. The structure adopts the typical fold of a class III viral fusion protein and displays an extensive positively charged electrostatic potential pattern, which resembles the glycoprotein of Dhori virus and is consistent with our previous predictions. In addition, compared to other previously defined class III viral fusion proteins, the structures of all thogotovirus glycoproteins and homologs are more similar to herpes virus glycoprotein Bs than to the rhabdovirus G proteins. Thus, class III viral fusion proteins are quite diverse in structure, and sub-classes may have developed during evolution.