Gender, BMI, and Age-Related Variations in Lower Limb Alignment Parameters and CPAK Phenotypes in Chinese Patients with Knee Osteoarthritis.

OBJECTIVES: Research on the distribution of and the variation in coronal plane alignment of the knee (CPAK) in the Chinese osteoarthritis population is limited. We aimed to establish the CPAK classification based on the characteristics of lower limb alignment in the Chinese osteoarthritis population. We also investigated variations in lower limb alignment parameters and CPAK phenotypes based on gender, body mass index (BMI), and age. METHODS: A retrospective study was conducted on a total of 944 knees diagnosed with osteoarthritis in 479 patients from January 2017 to December 2023. A scatterplot was used to describe the distribution of the CPAK classification, and the differences in lower limb alignment parameters and the CPAK classification were compared across genders (male, female), ages (middle-aged/<65 years, elderly/≥65 years), and BMI categories (normal/<25 kg/m2, overweight and obese/≥25 kg/m2) using the chi-squared test or Fisher's exact test. RESULTS: The average arithmetic hip-knee-ankle angle and joint line obliquity (JLO) were -3.03° ± 5.69° and 174.45° ± 4.29°, respectively. There was a higher prevalence of constitutional varus alignment in males and the overweight or obese group, while constitutional valgus alignment was more common in females and the normal BMI group (p < 0.05). Additionally, females had a greater apex distal JLO than males (p < 0.05). There were no statistically significant differences in lower limb alignment parameters among different age groups (p > 0.05). Although there were variations in alignment parameters across different genders and BMI categories in the knee osteoarthritis population, the predominant CPAK classifications were type I (38.03%), followed by type II (20.02%) and type IV (17.06%). CONCLUSION: The most common CPAK types were I, II, and IV, and they were not influenced by gender, BMI, or age, indicating that the CPAK classification can reliably reflect constitutional alignment. A better understanding of native alignment variability can aid in providing patient-specific recommendations when considering orthopedic alignment strategies.

The presence of a sinus tract is associated with reinfection after two-stage revision surgery for prosthetic hip joint infection: a case-control study.

BACKGROUND: Reinfection rates after two-stage revision (TSR) for prosthetic joint infection (PJI) range from 7.9 to 14%. Many factors, including sinus tracts, are associated with reinfection after this procedure. This study aimed to delineate whether the presence of sinus tract could increase reinfection rate after TSR and to investigate other potential risk factors for reinfection after TSR. METHODS: We conducted a case-control study by retrospectively reviewing patients who underwent TSR for prosthetic hip joint infection from 2002 to 2022. The case group included patients who developed reinfection after TSR, while the control group consisted of patients who did not experience reinfection. PJI and reinfection after TSR were defined based on Delphi-based international consensus criteria. Patient demographics, past medical history, clinical manifestations, laboratory results, interval between stages, microbiological culture results were collected. Univariate analyses were utilized to assess the effect of sinus tract on reinfection and to identify other risk factors for reinfection after TSR. RESULTS: Six patients with reinfection after TSR were included as the case group and 32 patients without reinfection were in the control group. Significant difference was observed in percentage of patients with sinus tracts between the two groups (67% in the case group versus 19% in the control group, p = 0.031, OR = 8.7). Significant difference was also found in percentage of patients with positive cultures of synovial fluid and synovium harvested during the first-stage revision between the two groups (100% in the case group versus 50% in the control group, p = 0.030). Additionally, patients in the case group had a significantly higher C-reactive protein (CRP) level prior to the second stage revision than that of patients in the control group (8.80 mg/L versus 2.36 mg/L, p = 0.005), despite normal CRP levels in all patients. CONCLUSIONS: Our study revealed that the presence of sinus tracts could significantly increase risk of postoperative reinfection after TSR. Positive cultures during the first stage revision and elevated CRP level prior to the second stage revision could also increase the risk of reinfection after TSR. Further studies with a larger sample size are required. TRIAL REGISTRATION: Retrospectively registered.

Correlation analysis between potential hepatotoxicity and ingredient content of polygoni multiflori caulis.

Polygoni Multiflori Caulis (PMC) is commonly used in clinical practice. While the adverse reactions of Polygoni Multiflori Radix (RPM) are well-known, the potential adverse reactions of PMC are often neglected. This article aims to clarify the relationship between hepatotoxic components in PMC and its various producing areas. This study provides a qualitative and quantitative analysis of PMC from various regions, which can serve as a basis for safe usage.

Synergic effects and possible mechanism of emodin and stilbene glycosides on colorectal cancer.

BACKGROUND: Polygonum multiflorum (PM) is a core herb that enhances immunity. It can also detoxify, reduce swelling, and intercept malaria. Its main components, emodin (EMD) and 2,3,5,4'-Tetrahydroxy stilbene-2-O-ß-D-glucoside (stilbene glycoside, TSG), have good anti-cancer potential. PURPOSE: The study aims to investigate synergic effects of EMD and TSG on CRC and its possible mechanism. METHODS: Network pharmacology and bioinformatics were used to identify targets. HPLC was used to analyze the effective ingredients in PM and to determine the content of the main ingredients. HT-29 cells were used for in vitro experiments. Cell Counting Kit-8 (CCK8) and scratch test were used to detect the effects of various chemical components of PM on the proliferation and migration of HT-29 cells, and Western Bolt (WB) test was used to evaluate the effects of EMD and TSG on P53 pathway. In vivo experiments, the effects of EMD and TSG were evaluated by measuring tumor weight and tumor volume in CRC mice model and histological analysis were carried out with HE staining. The expressions of HSP90, P53, COX2, and ROS were detected by quantitative reverse transcription polymerase chain reaction (PCR), and IL-1ß, IL-4, IL-6, IL-10, TGF-ß and IFN-γ were detected by enzyme linked immunosorbent assay (ELISA). WB and Immunohistochemistry (IHC) were used to detect the expression of P53 related proteins. RESULTS: Network pharmacology showed PM closely related to colorectal cancer pathway and the core targets included STAT3 and P53; bioinformatics indicated P53 played an important role in the development and prognosis of CRC; chemical analysis showed identified and quantified gallic acid (GA), cis-TSG, trans-TSG, Emodin glucoside(EMDG), physcion glucoside (PHYG), EMD in PM; EMD induced apoptosis and TSG inhibited migration of HT-29 cells; EMD and TSG could coordinately shrink tumor size of CRC mice, elevate expressions of F4/80, decrease the content of IL-6 and TGF-ß, promote tumor oxidized and reduce expression of P53 and STAT3 in the tumor. CONCLUSIONS: In vitro experiments showed that TSG inhibited cancer cell migration and EMD induced apoptosis. EMD and TSG had synergic effects on CRC, whose possible mechanism might be to regulate the expression of cytokines and inhibit P53 pathway.

Structure and pharmacokinetics/pharmacodynamics of the anticoagulant tetradecasaccharide oHG-14 as an intrinsic tenase inhibitor.

The intrinsic tenase complex (iXase) is an attractive antithrombotic target to treat or prevent pathological thrombosis with negligible bleeding risk. Fucosylated glycosaminoglycan (FG) is a promising anticoagulant by inhibiting iXase. A depolymerized FG (dHG-5) as an anticoagulant has been approved for clinical trials. Given that dHG-5 is a multi-component drug candidate consisting of a homologous series of oligosaccharides, it is difficult to predict a clear pharmacokinetics. Here, as a major oligosaccharide component, the tetradecasaccharide (oHG-14) was purified from dHG-5 and its structure was defined as L-Fuc3S4S-α(1,3)-L-Δ4,5GlcA-α(1,3)-{D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ß(1,3)-}3-D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ol. oHG-14 showed potent iXase inhibitory activity in vitro and antithrombotic effect in vivo comparable to dHG-5. After single subcutaneous administration of oHG-14 at 8, 14.4 and 32.4 mg/kg to rats, the absolute bioavailability was 71.6 %-80.9 % determined by the validated bioanalytical methods. The maximum concentration (Cmax) was 3.73, 8.07, and 11.95 µg/mL, respectively, and the time reaching Cmax (Tmax) was about 1 h. oHG-14 was mainly excreted by kidney as the parent compound with the elimination kinetics of first-order linear model. Anticoagulant activity of oHG-14 was positively correlated with its concentration in rat plasma. The pharmacokinetics/pharmacodynamics (PK/PD) of oHG-14 is similar to that of dHG-5. This study could provide supportive data for the clinical trial of dHG-5 and further development of pure oligosaccharide as an antithrombotic drug candidate.

A Cullin 5-based complex serves as an essential modulator of ORF9b stability in SARS-CoV-2 replication.

The ORF9b protein, derived from the nucleocapsid's open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.

Effects of Different Storage Times on the Stability of 12 Traditional Chinese Medicine Decoction Pieces.

As storage time increases, the quality of traditional Chinese medicines (TCMs) may change, and stability is an essential aspect of ensuring the safety and efficacy of TCMs. In this study, the effects of different storage times on the stability of 12 decoction pieces were evaluated. High-performance liquid chromatography was used to determine the contents of the active components in the 12 decoction pieces. The chemical composition data were analyzed using fingerprinting and clustering heatmap (CH). Results showed that during storage, significant variations (relative standard deviation > 10%) were observed in the levels of paeoniflorin in Paeoniae Radix Alba and Paeoniae Radix Rubra, hesperidin in Citri Reticulatae Pericarpium and Citri Reticulatae Pericarpium Viride, bufothionine in Siccus Bufo and chlorogenic acid in White Chrysanthemi Flos and Lonice Raejaponicae Caulis. However, calycosin-7-glucoside and calycosin in Astragali Radix Praeparata Cum Melle and chlorogenic acid in Lonicerae Japonicae Flos, Yellow Chrysanthemi Flos and Mori Folium were all <10%, which is consistent with the CH. Decoction pieces can be stored for up to six months, but it is recommended that volatile oil-containing and animal-based decoction pieces should not be stored for more than one month. This study provides new perspectives for the stability and quality control studies of TCM.

Targeting epidermal growth factor receptor signalling pathway: A promising therapeutic option for COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously producing new variants, necessitating effective therapeutics. Patients are not only confronted by the immediate symptoms of infection but also by the long-term health issues linked to long COVID-19. Activation of epidermal growth factor receptor (EGFR) signalling during SARS-CoV-2 infection promotes virus propagation, mucus hyperproduction, and pulmonary fibrosis, and suppresses the host's antiviral response. Over the long term, EGFR activation in COVID-19, particularly in COVID-19-induced pulmonary fibrosis, may be linked to the development of lung cancer. In this review, we have summarised the significance of EGFR signalling in the context of SARS-CoV-2 infection. We also discussed the targeting of EGFR signalling as a promising strategy for COVID-19 treatment and highlighted erlotinib as a superior option among EGFR inhibitors. Erlotinib effectively blocks EGFR and AAK1, thereby preventing SARS-CoV-2 replication, reducing mucus hyperproduction, TNF-α expression, and enhancing the host's antiviral response. Nevertheless, to evaluate the antiviral efficacy of erlotinib, relevant clinical trials involving an appropriate patient population should be designed.

TET2 is recruited by CREB to promote Cebpb, Cebpa, and Pparg transcription by facilitating hydroxymethylation during adipocyte differentiation.

Ten-eleven translocation proteins (TETs) are dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an important epigenetic mark that regulates gene expression during development and differentiation. Here, we found that the TET2 expression was positively associated with adipogenesis. Further, in vitro and in vivo experiments showed that TET2 deficiency blocked adipogenesis by inhibiting the expression of the key transcription factors CCAAT/enhancer-binding protein beta (C/EBPß), C/EBPα and peroxisome proliferator-activated receptor gamma (PPARγ). In addition, TET2 promoted 5hmC on the CpG islands (CGIs) of Cebpb, Cebpa and Pparg at the initial time point of their transcription, which requires the cAMP-responsive element-binding protein (CREB). At last, specific knockout of Tet2 in preadipocytes enabled mice to resist obesity and attenuated the obesity-associated insulin resistance. Together, TET2 is recruited by CREB to promote the expression of Cebpb, Cebpa and Pparg via 5hmC during adipogenesis and may be a potential therapeutic target for obesity and insulin resistance.

A turn-on fluorescence strategy for hypochlorous acid detection based on DNAzyme-assisted cyclic signal amplification.

Hypochlorous acid (HClO) is a crucial active oxygen component and one of the innate immunity's barrier substances in the body. Abnormal fluctuations in HClO concentration can lead to increased oxidative stress, cellular dysfunction, and the onset of various diseases. Thus, developing convenient, affordable, efficient, and sensitive methods to monitor HClO concentration in healthcare and pathophysiology research is highly significant. In this study, we developed a novel fluorescence strategy for HClO detection based on nucleic acid oxidative cleavage and Pb2+-dependent DNAzyme. By introducing a phosphorothioate site in the hairpin-structured nucleic acid sequence, the nucleic acid probe specifically recognized HClO and underwent oxidative cleavage. Upon cleavage, the enzyme strand is liberated, forming DNAzyme. This DNAzyme then cleaves the substrate strand, liberating the initially quenched fluorescent dyes and generating a turn-on fluorescent signal. The enzyme strand produced by the oxidative cleavage of HClO can be repeatedly utilized, thus realizing the cyclic signal amplification to reduce background noise. We verified the detection mechanism of this strategy through stepwise fluorescence spectroscopy analysis and electrophoresis. Under optimal experimental conditions, the method achieved a detection limit of 5.38 nM and a linear range of 1 nM-800 nM. This method demonstrated exceptional performance in actual biological sample testing and presented an exciting opportunity for expanded utilization in clinical diagnosis and medical research.

Total Hip Arthroplasty in the Nonparalytic Limb of Residual Poliomyelitis Patients: A Propensity Score Matched Study.

OBJECTIVE: Poliomyelitis is a rare neuromuscular disease that can cause hip osteoarthritis on the contralateral side due to an abnormal mechanical weight-bearing state, making some residual poliomyelitis patients candidates for total hip arthroplasty (THA). The aim of this study was to investigate the clinical outcome of THA in the nonparalytic limbs of these patients compared with those of non-poliomyelitis patients. METHODS: Patients treated between January 2007 and May 2021 were retrospectively identified in a single center arthroplasty database. Eight residual poliomyelitis cases that met the inclusion criteria were matched to non-poliomyelitis cases in a ratio of 1:2 based on age, sex, body mass index (BMI), age-adjusted Charlson comorbidity index (aCCI), surgeon, and operation date. The hip function, health-related quality of life, radiographic outcomes, and complications were analyzed with unpaired Student's t test, Mann-Whitney test, Fisher's exact test or analysis of covariance (ANCOVA). Survivorship analysis was determined using the Kaplan-Meier estimator analysis and Gehan-Breslow-Wilcoxon test. RESULTS: After a mean follow-up of about 5 years, patients with residual poliomyelitis had worse postoperative mobility outcomes(P < 0.05), but there was no difference in total modified Harris hip score (mHHS) or European quality of life-visual analogue scale (EQ-VAS) between the two groups (P > 0.05). There was no difference in radiographic outcomes or complications between the two groups, and patients had similar postoperative satisfaction (P > 0.05). No readmission or reoperation occurred in the poliomyelitis group (P > 0.05), but the postoperative limb length discrepancy (LLD) in the residual poliomyelitis group was greater than that in the control group (P < 0.05). CONCLUSION: Functional outcomes, health-related quality of life improvement were similarly significantly improved in the nonparalytic limb of residual poliomyelitis patients after THA compared with conventional osteoarthritis patients. However, the residual LLD and weak muscle strength of the affected side will still influence mobility, so residual poliomyelitis patients should be fully informed of this outcome before surgery.

A glucose-blue light AND gate-controlled chemi-optogenetic cell-implanted therapy for treating type-1 diabetes in mice.

Exogenous insulin therapy is the mainstay treatment for Type-1 diabetes (T1D) caused by insulin deficiency. A fine-tuned insulin supply system is important to maintain the glucose homeostasis. In this study, we present a designed cell system that produces insulin under an AND gate control, which is triggered only in the presence of both high glucose and blue light illumination. The glucose-sensitive GIP promoter induces the expression of GI-Gal4 protein, which forms a complex with LOV-VP16 in the presence of blue light. The GI-Gal4:LOV-VP16 complex then promotes the expression of UAS-promoter-driven insulin. We transfected these components into HEK293T cells, and demonstrated the insulin was secreted under the AND gate control. Furthermore, we showed the capacity of the engineered cells to improve the blood glucose homeostasis through implantation subcutaneously into Type-1 diabetes mice.

Gallic acid ameliorates dextran sulfate sodium-induced ulcerative colitis in mice via inhibiting NLRP3 inflammasome.

Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory bowel disease (IBD). The conventional drugs for UC may induce severe side effects. Herbal medicine is considered as a complementary and alternative choice for UC. Purpose: This study aims to estimate the effect of natural polyphenol gallic acid (GA) on the NLRP3 inflammasome with dextran sulfate sodium (DSS)-induced colitis in mice. Study design: The body weights and symptoms of BALB/c mice were recorded. Histological evaluation, ELISA, q-PCR, immunohistochemistry, and western blotting were carried out to observe the morphology, cytokine contents, mRNA expressions, and protein expressions, respectively. Lipopolysaccharide (LPS)-induced RAW264.7 macrophage was used to probe GA's effect on relative protein expression. Results: GA attenuated weight loss (p < 0.05), relieved symptoms, and ameliorated colonic morphological injury (p < 0.05) in mice with colitis induced by DSS. GA also lowered the contents of TNF-α, IL-1ß, IL-18, IL-33, and IFN-γ in the serum and colon of mice, which were elevated by DSS, downregulated protein, and mRNA expressions of the NLRP3 pathway in the colon tissue. Furthermore, GA downregulated the expressions of NLRP3 (p < 0.05), iNOS (p < 0.01), COX2 (p < 0.01), and P-p65 (p < 0.05), and suppressed NO release (p < 0.001) in LPS-induced RAW264.7 cells. Conclusion: GA ameliorated DSS-induced UC in mice via inhibiting the NLRP3 inflammasome. These findings furnish evidence for the anti-inflammatory effect of herbal medicines containing GA on UC.

Computer Navigation Assisted Restricted Kinematic Alignment Improves Short-Term Outcomes in Total Knee Arthroplasty: An Ambispective Cohort Study.

OBJECTIVES: Mechanical alignment (MA)-total knee arthroplasty (TKA) has been challenged due to the excessive soft tissue release and the evidence of the clinical outcomes of computer assisted navigation is still limited. The aim of this ambispective cohort study was to: (i) investigate whether computer assisted navigation is capable to achieve restricted kinematic alignment (rKA)-TKA; and (ii) compare the short-term outcomes between rKA-TKA and MA-TKA. METHODS: We retrospectively included 41 patients diagnosed with osteoarthritis who received MA-TKA between April 2019 and January 2021 and 43 patients diagnosed with osteoarthritis who received rKA-TKA were included in the prospective cohort from January 2021 to September 2021. Demographical, peri-operative, and radiological data were collected and compared. Unpaired two-sample t-test for continuous variables and χ2 test for categorical variables were used to compare various measurements in two groups. The patient-reported outcome measures at baseline, 10 days (T1), and 6 months (T6) after surgery were statistically analyzed by generalized estimating equation (GEE) models. RESULTS: Fourty-one patients (45 knees) and 43 patients (48 knees) were included in the MA and the rKA group respectively. Three constitutional knee phenotypes (II, I, IV) were the commonest in our population. Navigation improved the surgical accuracy (1.5° vs 3.5°, p < 0.001) and precision (interquartile range 4.0 vs 2.0, p < 0.001) in the rKA group than the MA group. The changes in Knee Injury and Osteoarthritis Outcome Score 12 (KOOS12), EuroQol five-dimension questionnaire (EQ5D) from baseline to T1 and T6 for patients with on-target rKA were larger than on-target MA counterparts (26.053 vs 18.607, P < 0.001(KOOS12, T1) , 0.457 vs 0.367 p < 0.001(EQ5D, T1) ; 51.017 vs 46.896, P = 0.023(KOOS12, T6) , 0.606 vs 0.565, P = 0.01(EQ5D, T6) ). Patients with on-target rKA had better Forgotten Joint Score (FJS) at T1 (54.126 vs 40.965, P = 0.002) compared with on-target MA counterparts. CONCLUSIONS: Computer assisted navigation achieved the level of accuracy required by rKA-TKA. rKA-TKA offered significantly better short-term outcomes than MA-TKA.

Total Knee Arthroplasty in Patients with Primary Sjögren's Syndrome: A Retrospective Case-Control Study Matched Patients without Rheumatic Diseases.

Background: The number of patients with primary Sjögren's syndrome (pSS) who require total knee arthroplasty (TKA) is expected to increase, and there are few studies describing their outcomes. This research was focused on the evaluation of a TKA cohort in pSS patients and to compare outcomes with those of matched individuals from the general population. Methods: From 2004 to 2020, we found 36 TKAs in 30 patients with pSS from the single-institution arthroplasty registry, and they were matched for age, gender, bilateral or unilateral surgery, American Society of Anesthesiologists (ASA) score, and year of surgery with 72 TKAs in 60 osteoarthritis patients without rheumatic diseases (1:2 ratio). Perioperative outcomes were obtained, and clinical evaluations were performed at the last follow-up. Results: After a mean six-year follow-up, both cohorts had similar knee function and health-related quality of life outcomes. The pSS group had more patients with post-operative anemia and hypoalbuminemia and more patients needing platelet transfusion. There were no significant differences in other complications, the rates of 90-day readmission, or overall revision. By multivariate analysis, the influencing factor for anemia in pSS patients was lower preoperative hemoglobin (OR = 0.334, 95% CI (0.125−0.889), p < 0.05). Conclusions: Our study demonstrated that pSS patients who received TKA could achieve comparable clinical outcomes to the general population. However, more attention should be paid to the perioperative hematological management of pSS patients who underwent TKA.

Recent Advances in Detection for Breast-Cancer-Derived Exosomes.

Breast cancer is the most common malignant tumor in women, its incidence is secret, and more than half of the patients are diagnosed in the middle and advanced stages, so it is necessary to develop simple and efficient detection methods for breast cancer diagnosis to improve the survival rate and quality of life of breast cancer patients. Exosomes are extracellular vesicles secreted by all kinds of living cells, and play an important role in the occurrence and development of breast cancer and the formation of the tumor microenvironment. Exosomes, as biomarkers, are an important part of breast cancer fluid biopsy and have become ideal targets for the early diagnosis, curative effect evaluation, and clinical treatment of breast cancer. In this paper, several traditional exosome detection methods, including differential centrifugation and immunoaffinity capture, were summarized, focusing on the latest research progress in breast cancer exosome detection. It was summarized from the aspects of optics, electrochemistry, electrochemiluminescence and other aspects. This review is expected to provide valuable guidance for exosome detection of clinical breast cancer and the establishment of more reliable, efficient, simple and innovative methods for exosome detection of breast cancer in the future.

A Label-Free Fluorescence Aptasensor Based on G-Quadruplex/Thioflavin T Complex for the Detection of Trypsin.

A novel, label-free fluorescent assay has been developed for the detection of trypsin by using thioflavin T as a fluorescent probe. A specific DNA aptamer can be combined by adding cytochrome c. Trypsin hydrolyzes the cytochrome c into small peptide fragments, exposing the G-quadruplex part of DNA aptamer, which has a high affinity for thioflavin T, which then enhances the fluorescence intensity. In the absence of trypsin, the fluorescence intensity was inhibited as the combination of cytochrome c and the DNA aptamer impeded thioflavin T's binding. Thus, the fluorescent biosensor showed a linear relationship from 0.2 to 60 µg/mL with a detection limit of 0.2 µg/mL. Furthermore, the proposed method was also successfully employed for determining trypsin in biological samples. This method is simple, rapid, cheap, and selective and possesses great potential for the detection of trypsin in bioanalytical and biological samples and medical diagnoses.

UBR5 Acts as an Antiviral Host Factor against MERS-CoV via Promoting Ubiquitination and Degradation of ORF4b.

Within the past 2 decades, three highly pathogenic human coronaviruses have emerged, namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The health threats and economic burden posed by these tremendously severe coronaviruses have paved the way for research on their etiology, pathogenesis, and treatment. Compared to SARS-CoV and SARS-CoV-2, MERS-CoV genome encoded fewer accessory proteins, among which the ORF4b protein had anti-immunity ability in both the cytoplasm and nucleus. Our work for the first time revealed that ORF4b protein was unstable in the host cells and could be degraded by the ubiquitin proteasome system. After extensive screenings, it was found that UBR5 (ubiquitin protein ligase E3 component N-recognin 5), a member of the HECT E3 ubiquitin ligases, specifically regulated the ubiquitination and degradation of ORF4b. Similar to ORF4b, UBR5 can also translocate into the nucleus through its nuclear localization signal, enabling it to regulate ORF4b stability in both the cytoplasm and nucleus. Through further experiments, lysine 36 was identified as the ubiquitination site on the ORF4b protein, and this residue was highly conserved in various MERS-CoV strains isolated from different regions. When UBR5 was knocked down, the ability of ORF4b to suppress innate immunity was enhanced and MERS-CoV replication was stronger. As an anti-MERS-CoV host protein, UBR5 targets and degrades ORF4b protein through the ubiquitin proteasome system, thereby attenuating the anti-immunity ability of ORF4b and ultimately inhibiting MERS-CoV immune escape, which is a novel antagonistic mechanism of the host against MERS-CoV infection. IMPORTANCE ORF4b was an accessory protein unique to MERS-CoV and was not present in SARS-CoV and SARS-CoV-2 which can also cause severe respiratory disease. Moreover, ORF4b inhibited the production of antiviral cytokines in both the cytoplasm and the nucleus, which was likely to be associated with the high lethality of MERS-CoV. However, whether the host proteins regulate the function of ORF4b is unknown. Our study first determined that UBR5, a host E3 ligase, was a potential host anti-MERS-CoV protein that could reduce the protein level of ORF4b and diminish its anti-immunity ability by inducing ubiquitination and degradation. Based on the discovery of ORF4b-UBR5, a critical molecular target, further increasing the degradation of ORF4b caused by UBR5 could provide a new strategy for the clinical development of drugs for MERS-CoV.

Primary Sjögren's syndrome is not associated with poor outcomes after total hip arthroplasty: a retrospective case-control study with a matched cohort of osteoarthritis patients.

INTRODUCTION: The number of patients with primary Sjögren's syndrome (PSS) requiring total hip arthroplasty (THA) is expected to increase, but few studies have detailed their outcomes. The purpose of this study was to evaluate a THA cohort of patients with avascular necrosis of the femoral head (ANFH) who had PSS and to compare their outcomes with those of matched patients with osteoarthritis. METHOD: A case-control study using data from a single-institution arthroplasty registry was performed. Forty-two THAs in 32 patients undergoing THA with a diagnosis of PSS were identified and were matched with 84 THAs in 64 patients with osteoarthritis (1:2 ratio). Functional and health-related quality of life (QoL) evaluations were performed, and complications were recorded at the last follow-up. Logistic regression was used to determine factors associated with reaching the transfusion trigger of hemoglobin < 8 g/dL (TT8) in PSS patients. RESULTS: After a mean 5-year follow-up, both cohorts had similar hip function and health-related QoL outcomes. The incision complications and reaching TT8 were greater in the PSS group. No differences were observed in the rate of 90-day readmission, reoperation, or overall revision. By multivariate analysis, the influencing factors for reaching TT8 in PSS patients were lower preoperative hemoglobin (OR = 0.842, 95% CI [0.741-0.958], P < 0.05). CONCLUSION: Our study demonstrated PSS patients who received THA due to ANFH could achieve clinical outcomes similar to those of non-PSS patients. Improving preoperative Hb level can reduce the risk of transfusion. Key Points • THA significantly improved hip function and health-related quality of life in PSS patients with osteonecrosis of the femoral head. • Patients with PSS were more likely to reach the transfusion trigger and higher rates of incision complications after THA. • Improving preoperative Hb level can reduce the risk of transfusion for PSS patients who underwent THA.

M6A-mediated upregulation of circMDK promotes tumorigenesis and acts as a nanotherapeutic target in hepatocellular carcinoma.

BACKGROUND: Emerging evidence suggest the critical role of circular RNAs (circRNAs) in disease development especially in various cancers. However, the oncogenic role of circRNAs in hepatocellular carcinoma (HCC) is still largely unknown. METHODS: RNA sequencing was performed to identify significantly upregulated circRNAs in paired HCC tissues and non-tumor tissues. CCK-8 assay, colony formation, transwell, and xenograft mouse models were used to investigate the role of circRNAs in HCC proliferation and metastasis. Small interfering RNA (siRNA) was used to silence gene expression. RNA immunoprecipitation, biotin pull-down, RNA pull-down, luciferase reporter assay and western blot were used to explore the underlying molecular mechanisms. RESULTS: Hsa_circ_0095868, derived from exon 5 of the MDK gene (named circMDK), was identified as a new oncogenic circRNA that was significantly upregulated in HCC. The upregulation of circMDK was associated with the modification of N6-methyladenosine (m6A) and poor survival in HCC patients. Mechanistically, circMDK sponged miR-346 and miR-874-3p to upregulate ATG16L1 (Autophagy Related 16 Like 1), resulting to the activation of PI3K/AKT/mTOR signaling pathway to promote cell proliferation, migration and invasion. Poly (ß-amino esters) (PAEs) were synthesized to assist the delivery of circMDK siRNA (PAE-siRNA), which effectively inhibited tumor progression without obvious adverse effects in four liver tumor models including subcutaneous, metastatic, orthotopic and patient-derived xenograft (PDX) models. CONCLUSIONS: CircMDK could serve as a potential tumor biomarker that promotes the progression of HCC via the miR-346/874-3p-ATG16L1 axis. The PAE-based delivery of siRNA improved the stability and efficiency of siRNA targeting circMDK. The PAE-siRNA nanoparticles effectively inhibited HCC proliferation and metastasis in vivo. Our current findings offer a promising nanotherapeutic strategy for the treatment of HCC.