Targeting Smurf1 to block PDK1-Akt signaling in KRAS-mutated colorectal cancer.

The phosphoinositide 3-kinase (PI3K)-Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K-Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K-Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1-Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K-Akt pathway and KRAS mutant cancer therapy.

Detection of Cancer Marker Flap Endonuclease 1 Using One-Pot Transcription-Powered Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Signal Expansion.

As a critical functional protein in DNA replication and genome stability, flap endonuclease 1 (FEN1) has been considered a promising biomarker and druggable target for multiple cancers. We report here a transcription-powered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a signal expansion platform for rapid and sensitive detection of FEN1. In this method, the probe cleavage by FEN1 generated a free 5' flap single-stranded DNA which could hybridize with the single-stranded T7 promoter-bearing template and trigger the extension. Then, the CRISPR guide RNA (crRNA) transcribed from the extended template activated the collateral DNase activity of Cas12a, releasing the fluorophore from the quenched DNA signal probe to report the FEN1 detection result. The high specificity for FEN1 was validated by comparing with other repair-relevant proteins. The limit of detection (LOD) could be as low as 0.03 mU, which is sensitive enough to detect the FEN1 activity in biological samples. In addition, the inhibition assay of FEN1 was also successfully achieved with this platform, proving its potential in inhibitor screening. In summary, this study provides a novel biosensor for FEN1 activity analysis and provides new insights into the development of CRISPR-based biosensors for non-nucleic acid targets.

The association between time in the glucose target range and abnormal ankle-brachial index: a cross-sectional analysis.

BACKGROUND: Time in range (TIR), a novel proxy measure of glucose control, is found closely related to diabetic microangiopathy and some other chronic complications, but the correlation between TIR and lower limb angiopathy has not been studied yet. Our purpose is to explore the relationship between TIR and abnormal ankle-brachial index(ABI) in type 2 diabetes. METHODS: We retrospectively collected patients' information from the database and performed cross-sectional analysis. A total of 405 type 2 diabetes patients were enrolled in this study. ABI was measured and patients were stratified into low, normal, and high groups according to ≤ 0.9, > 0.9 and < 1.3, ≥ 1.3 ABI values. All patients underwent continuous glucose monitoring(CGM), and TIR was defined as the percentage of time in which glucose was in the range of 3.9-10 mmol/L during a 24-h period. Correlations between TIR and abnormal ABI were analyzed using Spearman analysis. And logistic regression was used to explore whether TIR is an independent risk factor for abnormal ABI. RESULTS: The overall prevalence of abnormal ABI was 20.2% (low 4.9% and high 15.3%). TIR was lower in patients with abnormal ABI values (P = 0.009). The prevalence of abnormal ABI decreased with increasing quartiles of TIR (P = 0.026). Abnormal ABI was negatively correlated with TIR and positively correlated with hypertension, age, diabetes duration, UREA, Scr, ACR, TAR, MBG, and M values (P < 0.05). The logistic regression revealed a significant association between TIR and abnormal ABI, while HbA1C and blood glucose variability measures had no explicit correlation with abnormal ABI. Additionally, there was a significant difference in LDL between the low and high ABI groups (P = 0.009), and in Scr between normal and low groups (P = 0.007). And there were significant differences in TIR (P = 0.003), age (P = 0.023), UREA (P = 0.006), ACR (P = 0.004), TAR (P = 0.015), and MBG (P = 0.014) between normal and high ABI groups, and in diabetes duration between both normal and low (P = 0.023) and normal and high (P = 0.006) groups. CONCLUSIONS: In type 2 diabetes patients, abnormal ABI is associated with lower TIR, and the correlation is stronger than that with HbA1C. Therefore, the role of TIR should be emphasized in the evaluation of lower limb vascular diseases.

Efficacy and safety profiles of dolutegravir plus lamivudine vs . bictegravir/emtricitabine/tenofovir alafenamide in therapy-naïve adults with HIV-1.

BACKGROUND: Dual regimen dolutegravir (DTG) plus lamivudine (3TC) has demonstrated non-inferior efficacy compared to DTG-based three-drug regimens (3DRs), yet directly comparative data regarding the efficacy and safety of DTG + 3TC and bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) for therapy-naïve people with human immunodeficiency virus (HIV)-1 (PWH) are still limited. We aimed to assess the antiviral potency and safety profiles of DTG + 3TC vs. B/F/TAF based on antiretroviral therapy (ART)-naïve PWH in China. METHODS: This retrospective multicenter study enrolled PWH initiating ART with DTG + 3TC or B/F/TAF from 2020 to 2022 in Guangdong and Guangxi. We analyzed response rates based on target not detected (TND) status using intention-to-treat (ITT) analysis. Subgroups were formed based on baseline viral load (VL) (<100,000 vs . ≥100,000 copies/mL) and CD4 + cell count (<200 vs . ≥200 cell/µL). Median time to TND VL was assessed by Kaplan-Meier method. We also measured changes from baseline in CD4 + cell counts, CD4/CD8 ratio, lipid parameters, weight, creatinine (Cr), estimated glomerular filtration rate (eGFR), and drug-related adverse effects (DRAEs). RESULTS: We enrolled 280 participants, including 137 (48.9%) on DTG + 3TC and 143 (51.1%) on B/F/TAF. At week 48, 96.4% (132/137) on DTG+3TC and 100% (143/143) on B/F/TAF achieved TND ( P = 0.064). At week 12, TND responses were higher with B/F/TAF (78.3% [112/143]) than DTG+3TC (30.7% [42/137]) ( P <0.001). This trend held across subgroups. B/F/TAF achieved TND faster (12 weeks) than DTG+3TC (24 weeks) ( P <0.001). No differences were seen in CD4 + cell count and CD4/CD8 ratio, except in the high-VL subgroup, where B/F/TAF showed better recovery. DRAEs were significantly lower with B/F/TAF (4.9% [7/143]) than with DTG + 3TC (13.1% [18/137]) ( P = 0.016). Lipid parameters, body weight, and Cr increased in both groups over 48 weeks, with DTG+3TC showing a more favorable effect on triglycerides, high-density lipoprotein (HDL) cholesterol, and weight gain. CONCLUSIONS: In this real-life study, B/F/TAF led to a faster viral decline and fewer DRAEs compared to DTG+3TC. No significant difference was observed in the TND rate at week 48, regardless of baseline VL and CD4 + cell count. CD4 + recovery was superior for B/F/TAF in participants with high VL. The DTG + 3TC regimen had less impact on metabolic changes than B/F/TAF.

[Correlation between serum anti-P53 and familial clustering of hepatocellular carcinoma in Guangxi].

OBJECTIVE: To assess the correlation between familial clustering of hepatocellular carcinoma (HCC) and the level of anti-P53 in human serum in Guangxi. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect anti-P53 in 164 members from 20 HCC families and 164 members from non-cancer control families. Univariate analysis was performed to assess the correlation between seral level of P53 antibody and familial clustering of HCC. RESULTS: The level of P53 antibody was significantly higher in the members of HCC families than controls (Z=-3.04, P=0.002). After eliminating the interference of hepatitis B virus infection, this tendency still remains (P=0.011). And there was a significant difference between relatives of different degrees from HCC families (chi-square=11.593, P=0.021), with the expression of anti-P53 declining along with decrease in relationship coefficient. Furthermore, the number of individuals with high anti-P53 expression was also significantly greater in HCC families (95/164) than controls (71/164) (P=0.006). And the expression was rising along with the increasing HCC numbers (chi-square=16.068, P=0.000). Anti-P53 level was also greater in HCC families featuring sibling affection than parental affection (chi-square=12.679, P=0.002). Univariate analysis indicated that high expression of anti-P53 is a risk factor for development of HCC (OR=2.087, 95%CI: 1.270-3.431). CONCLUSION: High level of anti-P53 expression may be a factor for the clustering of HCC families in Guangxi, China.

Real-time fluorescence and colorimetric identification of bulbus fritillariae using recombinase assisted loop-mediated isothermal DNA amplification (RALA).

Bulbus Fritillariae (BF) is a kind of herbal medicine widely used in many countries including China, Japan, Korea, and so on. Among the known BF medicinal materials, Bulbus Fritillariae cirrhosae (BF cirrhosae) was reported to have the best curative effect. Due to the limited resources of BF cirrhosae, a lot of adulterants have emerged in the market, impairing the market order, resource development, and above all, clinical efficacy. Here, a novel nucleic acid amplification technique, Recombinase Assisted Loop-mediated isothermal DNA Amplification (RALA), was used to establish a real-time fluorescence isothermal molecular authentication method for five commonly used BF drugs. Moreover, this RALA-based assay can also be developed as a colorimetric detection method for on-site detection. Both real-time fluorescence and visual methods could detect as low as 0.1% genuine targets in the mixed samples. In summary, we report an isothermal detection system for five kinds of BF circulating in the market, providing a new choice for the molecular identification of BF drugs and showing promise in the laboratory testing as well as field identification of other herbal medicines.

Deubiquitylase OTUD6B stabilizes the mutated pVHL and suppresses cell migration in clear cell renal cell carcinoma.

Von Hippel-Lindau (VHL) is an important tumor suppressor, and its inactivation is a hallmark of inherited VHL disease and most sporadic clear cell renal cell carcinoma (ccRCC). VHL protein (pVHL) with missense point mutations are unstable and degraded by the proteasome because of the disruption of elongin binding. Deubiquitylase ovarian tumor domain-containing 6B (OTUD6B) had been documented to couple pVHL and elongin B to form stable VHL - elonginB - elonginC complex, which protects pVHL from degradation. However, whether OTUD6B governs the stability of pVHL wild type and the missense mutants in ccRCC remains largely elusive. Here, we reported that low OTUD6B level predicted poorer survival in ccRCC patients with VHL missense mutation, but not frameshift deletion and nonsense mutation. OTUD6B is able to interact with wild type pVHL and tumor-derived pVHL missense mutants, except for pVHL I151T, and decrease their ubiquitylation and proteasomal degradation in ccRCC cells. Functionally, we revealed that OTUD6B depletion enhanced cell migration and HIF-2α level in ccRCC cells in a pVHL dependent manner. In addition, OTUD6B depletion reduced the inhibitory effects of ectopic pVHL missense mutants on cell migration and HIF-2α level, except for pVHL I151T. Thus, we speculated that I151 residue might be one of key sites of pVHL binding to OTUD6B. These results suggested that OTUD6B is an important regulator for the stability of pVHL missense mutants, which provides a potential therapeutic strategy for ccRCC with VHL mutations.

Circulating osteocalcin is associated with time in range and other metrics assessed by continuous glucose monitoring in type 2 diabetes.

BACKGROUND: Osteocalcin, a protein secreted mainly by mature osteoblasts, has been shown to be involved in glucose metabolism through various pathways. However, few studies has explored the association between osteocalcin and Time in range (TIR). Continuous glucose monitoring (CGM) -derived metrics, such as TIR and other indexes have been gradually and widely used in clinical practice to assess glucose fluctuations. The main purpose of this study was to investigate the correlation between osteocalcin and indexes from CGM in patients with type 2 diabetes mellitus (T2DM). METHOD: The total number of 376 patients with T2D were enrolled, all of them performed three consecutive days of monitoring. They were divided into four groups on account of the quartile of osteocalcin. Time in range, Time below range (TBR), Time above range(TAR) and measures of glycemic variability (GV) were assessed for analysing. After a 100 g standard steamed bread meal, blood glucose (Glu0h Glu0.5 h, Glu1h, Glu2h, GLu3h), C-peptide (Cp0h, Cp0.5 h, Cp1h, Cp2h, Cp3h), serum insulin (INS0h, INS0.5 h, INS1h, INS2h, INS3h) concentrations at different time points were obtained. HOMA-IS, HOMA-ßwas calculated to evaluate insulin sensitivity and insulin secreting of the participants. RESULTS: Patients with higher osteocalcin level had higher TIR (P < 0.05). Spearman correlation analysis showed that osteocalcin was positively correlated with TBR (although the P value for TBR was greater than 0.05) (r = 0.227, P < 0.001 r = 0.068, P = 0.189) and negatively correlated with TAR (- 0.229, P < 0.001). Similarly, there was a negative correlation between osteocalcin and glycemic variability (GV) indicators, including SD, MBG, MODD, ADDR, and MAGE (P value of MAGE > 0.05). Multiple stepwise regression showed that osteocalcin was an independent contributor to TIR, TAR and HOMA-IS. CONCLUSION: Circulating osteocalcin is positively correlated with TIR and negatively correlated with MODD, ADDR, and MAGE. Osteocalcin may have a beneficial impact on glucose homeostasis in T2DM patients.

Ultrafast identification of Pinelliae Rhizoma using colorimetric direct-VPCR.

With the increasing growth of the herbal market, a rapid and easy-to-use system is highly desirable in the high-throughput identification of massive herbal medicine samples. Here, an ultrafast and colorimetric detection system was devised based on simplifying template preparation and a newly developed amplification technique, named colorimetric direct-VPCR. The system was successfully applied to the identification of Pinelliae Rhizoma. Compared to the traditional method, the whole test can be finished within 30 min from the sample treatment to the testing results. The method was evaluated by correctly identifying 72 samples obtained from 9 different habitats, demonstrating its high reliability. In summary, we present an ultrafast (less than 30 min) and colorimetric detection platform (under ultraviolet lamp) based on direct-VPCR for the identification of Pinelliae Rhizoma. The high practicability (100% accuracy) of this pipeline enables it to be a promising method in the routine detection of other herbal materials. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03035-9.

Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification.

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.

Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause acute respiratory distress syndrome, hypercoagulability, hypertension, and multiorgan dysfunction. Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis. In an analysis of a randomly collected cohort of 124 patients with COVID-19, we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity. By virtual screening of a U.S. FDA approved drug library, we identified an anticoagulation agent dipyridamole (DIP) in silico, which suppressed SARS-CoV-2 replication in vitro. In a proof-of-concept trial involving 31 patients with COVID-19, DIP supplementation was associated with significantly decreased concentrations of D-dimers (P < 0.05), increased lymphocyte and platelet recovery in the circulation, and markedly improved clinical outcomes in comparison to the control patients. In particular, all 8 of the DIP-treated severely ill patients showed remarkable improvement: 7 patients (87.5%) achieved clinical cure and were discharged from the hospitals while the remaining 1 patient (12.5%) was in clinical remission.

Elevated levels of von Willebrand factor and high mobility group box 1 (HMGB1) are associated with disease severity and clinical outcome of scrub typhus.

OBJECTIVES: This study aimed to investigate whether von Willebrand factor (vWF) and high mobility group box 1 (HMGB1) are associated with the severity and clinical outcome of scrub typhus and to seek novel biomarkers for surveillance and prediction of the prognosis of this infection. METHODS: Serum concentrations of vWF and HMGB1 were measured twice by ELISA for scrub typhus patients (n=103), once prior to doxycycline therapy and then on day 7 of doxycycline therapy; concentrations were measured once for healthy controls (n=32). RESULTS: Among the total 103 patients enrolled, 38 had disease complicated by multiple organ dysfunction syndrome (MODS). Serum concentrations of vWF and HMGB1 were significantly higher in all the patients than in the healthy controls, both prior to doxycycline treatment and on day 7 of doxycycline treatment (p<0.01). Furthermore, serum levels of vWF, HMGB1, and creatinine (SCr) in the patients with MODS increased distinctly, while the platelet (PLT) count diminished markedly compared to the levels in patients without MODS (p<0.01). The concentration of vWF was positively correlated with that of HMGB1 (r=0.764, p<0.001) and SCr (r=0.528, p<0.001), but negatively correlated with the PLT count (r=-0.632, p<0.001). Both HMGB1 and vWF were significantly associated with mortality in scrub typhus (area under the curve (AUC)=0.864, p=0.001, and AUC=0.862, p=0.001, respectively). CONCLUSIONS: Elevated levels of vWF and HMGB1 are associated with the severity and clinical outcome of scrub typhus. These represent possible new biomarkers for use in the assessment and prognostic prediction of this infection.