Design and synthesis of Thieno[3, 2-b]pyridinone derivatives exhibiting potent activities against Mycobacterium tuberculosis in vivo by targeting Enoyl-ACP reductase.

In this study, a series of novel thieno [3, 2-b]pyridinone derivatives were designed and synthesized using a scaffold hopping strategy. Six compounds showed potent anti-mycobacterial activity (minimum inhibitory concentration (MIC) ≤ 1 µg/mL) against Mycobacterium tuberculosis (Mtb) UAlRa. Compound 6c displayed good activity against Mtb UAlRv (MIC = 0.5-1 µg/mL). Compounds 6c and 6i also showed activity against Mtb UAlRa in macrophages and exhibited low cytotoxicity against LO-2 cells. The selected compounds displayed a narrow antibacterial spectrum, with no activity against representative Gram-positive, Gram-negative bacteria, as well as fungi. Furthermore, compound 6c demonstrated favorable oral pharmacokinetic properties with a T1/2 value of 47.99 h and exhibited good in vivo activity in an acute mouse model of tuberculosis (TB). The target of compound 6c was identified as a NADH-dependent enoyl-acyl carrier protein reductase (InhA) by genome sequencing of spontaneously compound 6c-resistant Mtb mutants, indicating that compound 6c may not require activation and can directly target InhA. In vitro antimicrobial assays against a recombinant M. smegmatis overexpressing the Mtb-InhA, along with InhA inhibition assays, confirmed that InhA is the target of thieno [3, 2-b]pyridinone derivatives. Overall, this study identified thieno [3, 2-b]pyridinone scaffold as a novel chemotype that is promising for the development of anti-TB agents.

Discovery and Validation of Ferroptosis-Associated Genes of Ulcerative Colitis.

Background: Ulcerative colitis (UC) is a long-lasting idiopathic condition, but its precise mechanisms remain unclear. Meanwhile, evidence has demonstrated that ferroptosis seems to interlock with the progress of UC. This research sought to identify hub genes of UC related to ferroptosis. Methods: First, the relevant profiles for this article were obtained from GEO database. From the FerrDb, 479 genes linked to ferroptosis were retrieved. Using analysis of the difference and WGCNA on colonic samples from GSE73661, the remaining six hub genes linked to ferroptosis and UC were discovered. Through logistic regression analyses, the diagnostic model was constructed and was then evaluated by external validation using dataset GSE92415. Afterwards, the correlation between immune cell filtration in UC and hub genes was examined. Finally, a mice model of colitis was established, and the results were verified using qRT-PCR. Results: We acquired six hub genes linked to ferroptosis and UC. In order to create a diagnostic model for UC, we used logistic regression analysis to screen three of the six ferroptosis related genes (HIF1A, SLC7A11, and LPIN1). The ROC curve showed that the three hub genes had outstanding potential for disease diagnosis (AUC = 0.976), which was subsequently validated in samples from GSE92415 (AUC = 0.962) and blood samples from GSE3365 (AUC = 0.847) and GSE94648 (AUC = 0.769). These genes might be crucial for UC immunity based upon the results on the immune system. Furthermore, mouse samples examined using qRT-PCR also verified our findings. Conclusion: In conclusion, the findings have important implications for ferroptosis and UC, and these hub genes may also offer fresh perspectives on the aetiology and therapeutic approaches of UC.

Indomethacin Does Not Reduce Post-ERCP Pancreatitis in High-Risk Patients Receiving Pancreatic Stenting.

BACKGROUND: Rectal indomethacin reduces pancreatitis following endoscopic retrograde cholangiopancreatography (ERCP). However, there is insufficient evidence regarding its added benefits in patients already receiving prophylactic pancreatic stenting. Our goal was to evaluate the impact of indomethacin in high-risk patients undergoing pancreatic stenting. METHODS: A cohort study was conducted on all patients who underwent the rescue cannulation technique for challenging bile duct cannulation (selected high-risk patients). Patients were split into two groups based on the prophylaxis method for post-ERCP pancreatitis (PEP): one receiving a combination of indomethacin and pancreatic stenting, while the other received pancreatic stenting alone. Comparative analyses were carried out on PEP, hyperamylasemia, gastrointestinal bleeding, and postoperative hospital stay among post-ERCP pancreatitis patients. RESULTS: Between November 2017 and May 2023, a total of 607 patients with native papillae were enrolled, with 140 grouped into the indomethacin plus stent group and 467 into the stent alone group. The overall PEP rate was 4.4% in the entire cohort, with no statistical differences observed between the groups in terms of PEP rates (P = 0.407), mild PEP (P = 0.340), moderate to severe PEP (P = 1.000), hyperamylasemia (P = 0.543), gastrointestinal bleeding (P = 0.392), and postoperative hospital stay (P = 0.521). Furthermore, sensitivity analysis using multivariable analysis also validated these findings. CONCLUSIONS: Indomethacin did not reduce the incidence or severity of PEP in high-risk patients who routinely received prophylactic pancreatic stent placement. Therefore, the additional administration of rectal indomethacin to further mitigate PEP appears to be not necessary.

Intranasal adenovirus-vectored Omicron vaccine induced nasal immunoglobulin A has superior neutralizing potency than serum antibodies.

The upper respiratory tract is the initial site of SARS-CoV-2 infection. Nasal spike-specific secretory immunoglobulin A (sIgA) correlates with protection against Omicron breakthrough infection. We report that intranasal vaccination using human adenovirus serotype 5 (Ad5) vectored Omicron spike in people who previously vaccinated with ancestral vaccine could induce robust neutralizing sIgA in the nasal passage. Nasal sIgA was predominantly present in dimeric and multimeric forms and accounted for nearly 40% of total proteins in nasal mucosal lining fluids (NMLFs). A low-level IgG could also be detected in NMLFs but not IgM, IgD, and IgE. After a complete nasal wash, sIgA in the nasal passage could be replenished rapidly within a few hours. A comparison of purified paired serum IgA, serum IgG, and nasal sIgA from the same individuals showed that sIgA was up to 3-logs more potent than serum antibodies in binding to spikes and in neutralizing Omicron subvariants. Serum IgG and IgA failed to neutralize XBB and BA.2.86, while nasal sIgA retained potent neutralization against these newly emerged variants. Further analysis showed that sIgA was more effective than IgG or IgA in blocking spike-mediated cell-to-cell transmission and protecting hACE2 mice from XBB challenge. Using a sIgA monoclonal antibody as a reference, we estimated that the total nasal sIgA contains about 2.6-3.9% spike-specific sIgA in NMLFs collected approximately one month after intranasal vaccination. Our study provided insights for developing intranasal vaccines that can induce sIgA to build an effective and mutation-resistant first-line immune barrier against constantly emerging variants.

The neurotransmitter calcitonin gene-related peptide shapes an immunosuppressive microenvironment in medullary thyroid cancer.

Neurotransmitters are key modulators in neuro-immune circuits and have been linked to tumor progression. Medullary thyroid cancer (MTC), an aggressive neuroendocrine tumor, expresses neurotransmitter calcitonin gene-related peptide (CGRP), is insensitive to chemo- and radiotherapies, and the effectiveness of immunotherapies remains unknown. Thus, a comprehensive analysis of the tumor microenvironment would facilitate effective therapies and provide evidence on CGRP's function outside the nervous system. Here, we compare the single-cell landscape of MTC and papillary thyroid cancer (PTC) and find that expression of CGRP in MTC is associated with dendritic cell (DC) abnormal development characterized by activation of cAMP related pathways and high levels of Kruppel Like Factor 2 (KLF2), correlated with an impaired activity of tumor infiltrating T cells. A CGRP receptor antagonist could offset CGRP detrimental impact on DC development in vitro. Our study provides insights of the MTC immunosuppressive microenvironment, and proposes CGRP receptor as a potential therapeutic target.

Measures of insulin resistance and beta cell function before and after treatment of HCV infection.

The association between chronic HCV infection and type 2 diabetes mellitus (T2DM) has been established; however, there is limited research on ß-cell function particularly in the pre-diabetic population. Here, we evaluated indices of ß-cell function and insulin sensitivity across the spectrum from normal glucose tolerance to T2DM in individuals with and without chronic hepatitis C (CHC), and the effects of antiviral treatments on these variables. A total of 153 non-cirrhotic, non-fibrotic CHC patients with a BMI <25 were enrolled in the study. Among them, 119 were successfully treated with either direct acting antiviral (DAA) drugs or pegylated interferon/ribavirin (IFN/RBV) anti-HCV therapy. Fasting state- and oral glucose tolerance test (OGTT)-derived indexes were used to evaluate ß-cell function and insulin sensitivity. Among all subjects, 19 (13%) had T2DM and 21% exhibited pre-diabetes including 8% isolated impaired fasting glucose (IFG) and 13% combined IFG and impaired glucose tolerance (IGT). Early and total insulin secretion adjusted for the degree of insulin resistance were decreased in pre-diabetic CHC patients compared to HCV-uninfected individuals. Viral eradication through DAA or IFN/RBV therapy demonstrated positive impacts on insulin sensitivity and ß-cell function in CHC patients who achieved sustained virologic response (SVR), regardless of fasting or OGTT state. These findings emphasize the role of HCV in the development of ß-cell dysfunction, while also suggesting that viral eradication can improve insulin secretion, reverse insulin resistance, and ameliorate glycemic control. These results have important implications for managing pre-diabetic CHC patients and could prevent diabetes-related clinical manifestations and complications.

Monitoring Cattle Ruminating Behavior Based on an Improved Keypoint Detection Model.

Cattle rumination behavior is strongly correlated with its health. Current methods often rely on manual observation or wearable devices to monitor ruminating behavior. However, the manual monitoring of cattle rumination is labor-intensive, and wearable devices often harm animals. Therefore, this study proposes a non-contact method for monitoring cattle rumination behavior, utilizing an improved YOLOv8-pose keypoint detection algorithm combined with multi-condition threshold peak detection to automatically identify chewing counts. First, we tracked and recorded the cattle's rumination behavior to build a dataset. Next, we used the improved model to capture keypoint information on the cattle. By constructing the rumination motion curve from the keypoint information and applying multi-condition threshold peak detection, we counted the chewing instances. Finally, we designed a comprehensive cattle rumination detection framework to track various rumination indicators, including chewing counts, rumination duration, and chewing frequency. In keypoint detection, our modified YOLOv8-pose achieved a 96% mAP, an improvement of 2.8%, with precision and recall increasing by 4.5% and 4.2%, enabling the more accurate capture of keypoint information. For rumination analysis, we tested ten video clips and compared the results with actual data. The experimental results showed an average chewing count error of 5.6% and a standard error of 2.23%, verifying the feasibility and effectiveness of using keypoint detection technology to analyze cattle rumination behavior. These physiological indicators of rumination behavior allow for the quicker detection of abnormalities in cattle's rumination activities, helping managers make informed decisions. Ultimately, the proposed method not only accurately monitors cattle rumination behavior but also provides technical support for precision management in animal husbandry, promoting the development of modern livestock farming.

Farnesyltransferase inhibitor lonafarnib suppresses respiratory syncytial virus infection by blocking conformational change of fusion glycoprotein.

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.

The impact of preoperative calcitonin screening on the prognosis of patients with medullary thyroid cancer: a retrospective multicenter cohort study.

PURPOSE: There is still controversy in different guidelines regarding the necessity of routine preoperative calcitonin (Ctn) testing in medullary thyroid cancer (MTC). The level of preoperative Ctn may influence the extent of surgery. METHODS: This retrospective multicenter cohort study involved 149 MTC patients from 6 centers between 2013 to 2023. Clinical characteristics, surgical procedure and clinical outcomes were compared between Ctn-screened and Non-screened group. Kaplan-Meier method was used to estimate recurrence-free survival (RFS) and overall survival (OS). RESULTS: In total, 127 MTC patients with preoperative Ctn screening and 22 MTC patients without screening were analyzed. MTC patients with preoperative Ctn screening underwent more radical surgical procedures including total thyroidectomy and lymph node dissection, compared to those without screening (84.3% vs. 68.2% and 91.3% vs. 72.7%, respectively). The rate of recurrence and death were lower in the Ctn-screened group (16.1% vs. 36.4%, 0.8% vs. 18.2%, respectively). The survival curve showed a significantly better overall survival in Ctn-screened group than Non-screened group (HR:17.932, 95% CI 1.888-170.294, p-value = 0.001), while no significant difference was observed of RFS between two groups (HR:1.6, 95% CI 0.645-3.966, p-value = 0.307). CONCLUSION: Preoperative Ctn screening can prompt surgeons choosing more radical initial surgical treatment for MTC patients, potentially leading to better long-term outcomes. Further evaluation of the cost-effectiveness of routine Ctn screening in thyroid nodule patients is warranted.

Cryo-EM structures of Thogoto virus polymerase reveal unique RNA transcription and replication mechanisms among orthomyxoviruses.

Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5' end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses' broad-infectivity range.

Transpancreatic Sphincterotomy After Double Guidewire Technique Was Noninferior to Primary Transpancreatic Sphincterotomy in Difficult Biliary Cannulation.

BACKGROUND: When unintentional pancreatic duct access occurs during difficult biliary cannulation, the double guidewire (DGW) or transpancreatic sphincterotomy (TPS) may be utilized. DGW can be easily switched to TPS due to the existing guidewire in the pancreatic duct. However, the efficacy of TPS after DGW, named sequential DGW-TPS technique, versus primary TPS has not been assessed. AIMS: Our aim was to compare the benefits and adverse events of sequential DGW-TPS technique and primary TPS. METHODS: We performed a comparative retrospective cohort study that enrolled a total of 117 patients with native papillae. The patients were divided into one of 2 groups according to the primary bile duct access technique (sequential DGW-TPS or primary TPS), both with pancreatic stenting. RESULTS: Between November 2017 and May 2023, a total of 84 patients were grouped into sequential DGW-TPS and 33 into primary TPS. The overall post-ERCP pancreatitis (PEP) rate was 4.3% in the entire cohort, with no statistical differences were observed between the groups in terms of PEP rates (P = 0.927), PEP severity (P = 1.000), first biliary cannulation success (P = 0.621), overall cannulation success (P = 1.000), hyperamylasemia incidence (P = 0.241), elevated amylase levels (P = 0.881), and postoperative hospital stay (P = 0.185). Furthermore, these results remained consistent in multivariable regression analysis. CONCLUSIONS: The sequential DGW-TPS technique showed a comparable safety and biliary cannulation success rate to primary TPS in difficult biliary cannulation. Given the potential long-term complications associated with TPS, DGW should be first if inadvertent pancreatic access occurs, with TPS serving as second only if DGW fails.

Interlaminar Shear Strength Change and Storage Life Prediction of Carbon Fiber/Epoxy Composites with Hygrothermal Accelerated Aging.

In order to investigate the durability of fiber-reinforced polymer composites in hygrothermal environments, hygrothermal accelerate aging tests, for 360 days at 70 °C, RH70%; 70 °C, RH85%; 85 °C, RH70%; and 85 °C, RH85% and natural storage for 2 years in Guangzhou, China, were carried out for composite laminates. Then, the moisture absorption and interlaminar shear strength were measured. The hygrothermal damage mechanism of the composite was studied by Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and field emission scanning electron microscopy (FSEM). A dual stress storage life prediction model and the equivalent relationship between natural storage and hygrothermal acceleration were established. The results show that the order of moisture absorption rates, moisture absorption contents, and the severity effect order on the interlaminar shear strength is RH85%; 85 °C > 70 °C; RH85% > 85 °C; RH70% > 70 °C; and RH70%. The time to achieve an effective moisture absorption balance is opposite to this. The moisture absorption rate meets Fick's law before the effective moisture absorption balance, and then shows a linear trend. The interlayer shear strength still decreases exponentially with aging, which is mainly caused by the resin plasticization and interface weakening. Hygrothermal accelerated aging for 13.4831 days at 85 °C; RH85% is equivalent to that for one-year actual storage in Guangzhou. According to the failure criterion of shear strength decreasing to 77%, the storage life of T700/epoxy in Guangzhou is 14.4661 years.

Efficacy and safety of GST-HG171 in adult patients with mild to moderate COVID-19: a randomised, double-blind, placebo-controlled phase 2/3 trial.

Background: GST-HG171 is a potent, broad-spectrum, orally bioavailable small-molecule 3C like protease inhibitor that has demonstrated greater potency and efficacy compared to Nirmatrelvir in pre-clinical studies. We aimed to evaluate the efficacy and safety of orally administered GST-HG171 plus Ritonavir in patients with coronavirus disease 2019 (COVID-19) infected with emerging XBB and non-XBB variants. Methods: This randomised, double-blind, placebo-controlled phase 2/3 trial was conducted in 47 sites in China among adult patients with mild-to-moderate COVID-19 with symptoms onset ≤72 h. Eligible patients were randomised 1:1 to receive GST-HG171 (150 mg) plus Ritonavir (100 mg) or corresponding placebo tablets twice daily for 5 days, with stratification factors including the risk level of disease progression and vaccination status. The primary efficacy endpoint was time to sustained recovery of clinical symptoms within 28 days, defined as a score of 0 for 11 COVID-19-related target symptoms for 2 consecutive days, assessed in the modified intention-to-treat (mITT) population. This trial was registered at ClinicalTrials.gov (NCT05656443) and Chinese Clinical Trial Registry (ChiCTR2200067088). Findings: Between Dec 19, 2022, and May 4, 2023, 1525 patients were screened. Among 1246 patients who underwent randomisation, most completed basic (21.2%) or booster (74.9%) COVID-19 immunization, and most had a low risk of disease progression at baseline. 610 of 617 who received GST-HG171 plus Ritonavir and 603 of 610 who received placebo were included in the mITT population. Patients who received GST-HG171 plus Ritonavir showed shortened median time to sustained recovery of clinical symptoms compared to the placebo group (13.0 days [95.45% confidence interval 12.0-15.0] vs. 15.0 days [14.0-15.0], P = 0.031). Consistent results were observed in both SARS-CoV-2 XBB (45.7%, 481/1053 of mITT population) and non-XBB variants (54.3%, 572/1053 of mITT population) subgroups. Incidence of adverse events was similar in the GST-HG171 plus Ritonavir (320/617, 51.9%) and placebo group (298/610, 48.9%). The most common adverse events in both placebo and treatment groups were hypertriglyceridaemia (10.0% vs. 14.7%). No deaths occurred. Interpretation: Treatment with GST-HG171 plus Ritonavir has demonstrated benefits in symptom recovery and viral clearance among low-risk vaccinated adult patients with COVID-19, without apparent safety concerns. As most patients were treated within 2 days after symptom onset in our study, confirming the potential benefits of symptom recovery for patients with a longer duration between symptom onset and treatment initiation will require real-world studies. Funding: Fujian Akeylink Biotechnology Co., Ltd.

Preclinical evaluation of the SARS-CoV-2 Mpro inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir.

Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (Mpro), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from Mpro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 Mpro:RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named 'leritrelvir' for COVID-19 treatment in China.

Recent advances in CRISPR-Cas9-based genome insertion technologies.

Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.

Clinical prediction models for cervical lymph node metastasis of papillary thyroid carcinoma.

PURPOSE: Accurate preoperative diagnosis of lymph node metastasis (LNM) in papillary thyroid carcinoma (PTC) remains an unsolved problem. This study aimed to construct a nomogram and scoring system for predicting LNM based on the clinical characteristics of patients with PTC. METHODS: 1400 patients with PTC who underwent thyroidectomy and lymph node dissection at the First Affiliated Hospital of Sun Yat-sen University were retrospectively enrolled and randomly divided into training and internal testing sets. Furthermore, 692 patients with PTC from three other medical centers were collected as external testing sets. Least absolute shrinkage and selection operator (LASSO) was used to screen the predictors, and a nomogram was constructed. In addition, a scoring system was constructed using 10-fold cross-validation. The performances of the two models were verified among datasets and compared with preoperative ultrasound (US). RESULTS: Six independent predictors were included in the multivariate logistic model: age, sex, US diagnosis of LNM, tumor diameter, location, and thyroid peroxidase antibody level. The areas under the receiver operating characteristic curve (AUROC) (95% confidence interval) of this nomogram in the training, internal testing, and three external testing sets were 0.816 (0.791-0.840), 0.782 (0.727-0.837), 0.759 (0.699-0.819), 0.749 (0.667-0.831), and 0.777 (0.726-0.828), respectively. The AUROC of the scoring system were 0.810 (0.785-0.835), 0.772 (0.718-0.826), 0.736 (0.675-0.798), 0.717 (0.635-0.799) and 0.756 (0.704-0.808), respectively. The prediction performances were both significantly superior to those of preoperative US (P < 0.001). CONCLUSION: The nomogram and scoring system performed well in different datasets and significantly improved the preoperative prediction of LNM than US alone.

[An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay].

OBJECTIVE: To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay. METHODS: Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay. RESULTS: The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR. CONCLUSION: The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

Structure-based discovery of dual pathway inhibitors for SARS-CoV-2 entry.

Since 2019, SARS-CoV-2 has evolved rapidly and gained resistance to multiple therapeutics targeting the virus. Development of host-directed antivirals offers broad-spectrum intervention against different variants of concern. Host proteases, TMPRSS2 and CTSL/CTSB cleave the SARS-CoV-2 spike to play a crucial role in the two alternative pathways of viral entry and are characterized as promising pharmacological targets. Here, we identify compounds that show potent inhibition of these proteases and determine their complex structures with their respective targets. Furthermore, we show that applying inhibitors simultaneously that block both entry pathways has a synergistic antiviral effect. Notably, we devise a bispecific compound, 212-148, exhibiting the dual-inhibition ability of both TMPRSS2 and CTSL/CTSB, and demonstrate antiviral activity against various SARS-CoV-2 variants with different viral entry profiles. Our findings offer an alternative approach for the discovery of SARS-CoV-2 antivirals, as well as application for broad-spectrum treatment of viral pathogenic infections with similar entry pathways.

Energy-economy-environment assessments of refrigerants R152a and R134a in a vapor compression refrigeration system using a variable displacement oil-free linear compressor.

In light of the adverse environmental impact of the R134a refrigerant, replacing it with a more environmentally friendly refrigerant has become imperative than ever. This study presents an experimental investigation into the utilization of R152a and R134a refrigerants in a vapor compression refrigeration system employing a variable displacement oil-free linear compressor. The potential for the replacement of R134a with R152a was examined based on energy, environmental, and economic performance analyses. The outcomes indicated that R152a exhibited a higher coefficient of performance (COP) in comparison to R134a under identical operating conditions. Specifically, when the pressure ratio was 2.0 and the piston stroke was 11 mm, R152a's COP was 13.0% higher than R134a. It was also discovered that reducing the operating stroke and increasing the pressure ratio could effectively lower CO2 emissions and total costs. Under the 2.0 pressure ratio and 9-mm piston stroke, R134a produced 1082.4 kg more CO2 emissions than R152a, representing a 209% increase. In addition, the R152a and R134a total cost was reduced by 8.3% with the 2.5 pressure ratio and 11-mm piston stroke. Notably, the results of the current study demonstrated that R152a outperformed R134a in energy consumption, environmental friendliness, and economy in oil-free linear compressor refrigeration systems. R152a used less electric power, generated fewer CO2 emissions, and naturally reduced predicted running costs in order to maintain the same COP.