Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

Prophylactic chemotherapeutic hyperthermic intraperitoneal perfusion reduces peritoneal metastasis in gastric cancer: a retrospective clinical study.

BACKGROUND: Peritoneal metastasis is the most frequent failure in gastric cancer. This study evaluated the role of prophylactic chemotherapeutic hyperthermic intraperitoneal perfusion (CHIP) in patients after D2 dissection. METHODS: Gastric cancer patients after D2 dissection were enrolled in this study. Patients received either chemotherapy (IV group) or CHIP (CHIP group). Sites of recurrence or metastasis, disease-free survival (DFS), overall survival (OS) and adverse events were evaluated. RESULTS: Twenty-two patients received CHIP treatment, and 21 patients received chemotherapy alone. The median DFS time was 24.5 and 36.5 months in the IV group and CHIP group (P = 0.044), respectively. The median OS time was 33.1 months in the IV group and not reached in the CHIP group (P = 0.037). We also found that CHIP could reduce the total recurrence/metastasis rate, especially that of peritoneal metastasis. In the subgroup analysis, DFS and OS were both superior in deficient mismatch repair (dMMR) patients than in proficient MMR (pMMR) patients. CONCLUSION: This hypothesis-generating study indicates that CHIP might be feasible for gastric cancer patients after D2 resection.

Enhanced visible light photocatalytic performance of carbon and oxygen co-doped carbon nitride with a three-dimensional structure: Performance and mechanism study.

As a cost-effective photocatalyst, carbon nitride (g-C3N4) holds tremendous promise for addressing energy shortages and environmental pollution. However, its application is limited by disadvantages such as low specific surface area and easy recombination of photogenerated electron-hole pairs. This study introduces C and O co-doped g-C3N4 with a three-dimensional (3D) structure achieved through a straightforward one-step calcination process, demonstrating excellent photocatalytic activity of hydrogen production and oxytetracycline degradation, with superoxide radicals as the primary active species. We propose a plausible enhanced mechanism based on systematic characterizations and density functional theory calculations. The 3D structure confers a substantial specific surface area, enhancing both the adsorption area and active sites of catalysts while bolstering structural stability. Co-doping optimizes the band structure and electric conductivity of the catalyst, facilitating rapid migration of photogenerated charges. The synergistic effects of these enhancements significantly elevate the photocatalytic performance. This study presents a convenient and feasible method for the preparation of dual-regulated photocatalysts with outstanding performance.

Identification and characterization of ABC proteins in an important rice insect pest, Cnaphalocrocis medinalis unveil their response to Cry1C toxin.

Rice leaffolder (Cnaphalocrocis medinalis) is an important insect pest in paddy fields. Due to their essential role in the physiology and insecticidal resistance, ATP-binding cassette (ABC) proteins were studied in many insects. In this study, we identified the ABC proteins in C. medinalis through genomic data and analyzed their molecular characteristics. A total of 37 sequences with nucleotide-binding domain (NBD) were identified as ABC proteins and belonged to eight families (ABCA-ABCH). Four structure styles of ABC proteins were found in C. medinalis, including full structure, half structure, single structure, and ABC2 structure. In addition to these structures, TMD-NBD-TMD, NBD-TMD-NBD, and NBD-TMD-NBD-NBD were found in C. medinalis ABC proteins. Docking studies suggested that in addition to the soluble ABC proteins, other ABC proteins including ABCC4, ABCH1, ABCG3, ABCB5, ABCG1, ABCC7, ABCB3, ABCA3, and ABCC5 binding with Cry1C had higher weighted scores. The upregulation of ABCB1 and downregulation of ABCB3, ABCC1, ABCC7, ABCG1, ABCG3, and ABCG6 were associated with the C. medinalis response to Cry1C toxin. Collectively, these results help elucidate the molecular characteristics of C. medinalis ABC proteins, pave the way for further functional studies of C. medinalis ABC proteins, including their interaction with Cry1C toxin, and provide potential insecticide targets.

Mycobacterium tuberculosis PE_PGRS19 Induces Pyroptosis through a Non-Classical Caspase-11/GSDMD Pathway in Macrophages.

The PE/PPE protein family commonly exists in pathogenic species, such as Mycobacterium tuberculosis, suggesting a role in virulence and its maintenance. However, the exact role of most PE/PPE proteins in host-pathogen interactions remains unknown. Here, we constructed a recombinant Mycobacterium smegmatis expressing M. tuberculosis PE_PGRS19 (Ms_PE_PGRS19) and found that PE_PGRS19 overexpression resulted in accelerated bacterial growth in vitro, increased bacterial survival in macrophages, and enhanced cell damage capacity. Ms_PE_PGRS19 also induced the expression of pro-inflammatory cytokines, such as IL-6, TNF-α, IL-1ß, and IL-18. Furthermore, we demonstrated that Ms_PE_PGRS19 induced cell pyroptosis by cleaving caspase-11 via a non-classical pathway rather than caspase-1 activation and further inducing the cleavage of gasdermin D, which led to the release of IL-1ß and IL-18. To the best of our current knowledge, this is the first report of a PE/PPE family protein activating cell pyroptosis via a non-classical pathway, which expands the knowledge on PE/PPE protein functions, and these pathogenic factors involved in bacterial survival and spread could be potential drug targets for anti-tuberculosis therapy.

Lysophosphatidic Acid Improves Human Sperm Motility by Enhancing Glycolysis and Activating L-Type Calcium Channels.

Until now, the molecular mechanisms underlining sperm motility defect causing male infertility are still poorly understood. Safe and effective compounds or drugs that can improve sperm motility are also very limited. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid and a bioactive intermediate with multiple biological activities. It has been detected in various body fluids such as serum, plasma, saliva, tears, blister fluids, hen egg white, and ascites from patients with ovarian cancer. LPA is also abundant in seminal plasma and follicular fluid. It enhances follicle stimulation, improves oocyte fertilization, and promotes early embryonic development and embryo implantation. However, the physiological role of LPA in the male reproductive system remains unknown. Here, our study showed that LPA significantly improved the motility parameters of human sperm hyperactivation in a dose-dependent manner. The LPA-induced elevation of sperm motility is dependent on bovine serum albumin (BSA) but independent of the classical BSA-induced sAC/cAMP/PKA signaling pathway. The enhancement of sperm motility by LPA could not be blocked by CCCP, a respiratory inhibitor suppressing mitochondrial ATP production. Moreover, LPA improved the activity of triosephosphate isomerase in glycolysis. Meanwhile, LPA treatment significantly increased ATP and phosphoenolpyruvate levels and decreased ADP content during sperm glycolysis. Notably, none of known or identified LPA receptors was detected in human sperm. Further investigations showed that LPA promoted sperm motility through L-type calcium channels. In summary, this study revealed the involvement of LPA in the regulation for human sperm motility by enhancing glycolysis and activating L-type calcium channels. The current findings may shed new light on the understanding of causes of asthenozoospermia, and indicate that LPA could be used as a novel therapeutic agent to improve sperm function and fertilizing capacity.

[Construction and Application Optimization of the Chl-a Forecast Model ARIMA for Lake Taihu].

As an important indicator of phytoplankton biomass in lakes, the chlorophyll-a (Chl-a) concentration reflects the abundance and variation of phytoplankton in the water. Based on the monthly monitoring data of Chl-a and environmental factors in Lake Taihu from December 1999 to August 2019, key environmental factors related to Chl-a and their relationships were found using the principal component analysis (PCA) method. A multiple linear stepwise regression model and an auto-regressive integrated moving average (ARIMA) model were developed to predict the monthly Chl-a concentrations. The results showed that the Chl-a concentrations in Lake Taihu exhibited clear seasonal change characteristics and an overall trend of a gradual increase. The changes in total phosphorus (TP), the permanganate index, monthly average temperature (MAT), and monthly rainfall (MR) matched the Chl-a concentrations relatively well, whereas the changes in total nitrogen (TN) and ammonium nitrogen (NH4+-N) lagged significantly. The PCA results showed that the increased phytoplankton biomass and consequent algae outbreaks in Lake Taihu were not limited to the effect of a single factor such as TN or TP, but were comprehensively affected by multiple factors such as TN, NH4+-N, TP, the permanganate index, MR, and MAT. Through further validation, the ARIMA model of Chl-a concentrations was proved to be significantly better than the multiple linear stepwise regression model, especially when considering the key environmental factors as independent variables and optimizing their values. The established ARIMA (0,1,1) (0,1,1) model would be helpful for forecasting algae blooms in Lake Taihu and provide useful suggestions for water environmental management, such as water resources dispatch and regulation.

Dietary restriction and/or exercise training impairs spermatogenesis in normal rats.

Dietary restriction and/or exercise has been shown to have multiple benefits for health. However, its effects on reproductive health and the mechanisms by which it regulates reproductive function remain unclear. Here, to evaluate its effects on spermatogenesis and sperm function, rats were divided into 4 groups: ad libitum-fed sedentary control, dietary restriction (DR), exercise training (ET), and dietary restriction plus exercise training (DR+ET) groups. Results indicated that body weight, epididymal fat pad weight, and sperm counts were significantly reduced in the DR, ET, and DR+ET groups. Moreover, sperm motility and capacitation-associated protein tyrosine phosphorylation were suppressed in the DR and DR+ET groups, but not the ET group. Microarray analysis revealed that the number of downregulated genes was higher than that of upregulated genes in the DR and/or ET groups. About half of the downregulated genes are common after exercise training and/or diet restriction. Gene ontology analysis showed that downregulated genes in the DR, ET, and DR+ET groups affected spermatogenesis through overlapping pathways, including glucocorticoid, corticosteroid, extracellular structure organization, and estradiol responses. Our findings suggest that diet restriction and/or exercise training may present potential risks to male reproductive dysfunction by disrupting normal gene expression patterns in the testis. Novelty: Dietary restriction and/or exercise can lead to the damage of spermatogenesis as well as sperm maturation. Sperm functional changes are more sensitive to dietary restriction than exercise training. Dietary restriction and exercise impair spermatogenesis through overlapping biological pathways in the testis.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Dynamics Tracking.

Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples. Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients. Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections. Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.

Role of the PE/PPE Family in Host-Pathogen Interactions and Prospects for Anti-Tuberculosis Vaccine and Diagnostic Tool Design.

The pe/ppe genes are found in pathogenic, slow-growing Mycobacterium tuberculosis and other M. tuberculosis complex (MTBC) species. These genes are considered key factors in host-pathogen interactions. Although the function of most PE/PPE family proteins remains unclear, accumulating evidence suggests that this family is involved in M. tuberculosis infection. Here, we review the role of PE/PPE proteins, which are believed to be linked to the ESX system function. Further, we highlight the reported functions of PE/PPE proteins, including their roles in host cell interaction, immune response regulation, and cell fate determination during complex host-pathogen processes. Finally, we propose future directions for PE/PPE protein research and consider how the current knowledge might be applied to design more specific diagnostics and effective vaccines for global tuberculosis control.

A study of the white-coat phenomenon in patients with primary hypertension.

The objective of this study was to evaluate whether the discrepancy between clinic and home blood pressure (BP) in hypertensive subjects would disappear or diminish in magnitude if the BP measurement was taken under controlled conditions differing only with respect to location (clinic vs. home). Three hundred and sixty-seven patients aged 34-84 years with primary hypertension were enrolled. All of the patients or their spouses were taught to measure BP correctly with their own sphygmomanometer at home. The home BP value (HBP) was calculated as the average of 45 readings over 15 days. On days 6, 12, and 18 of the measurement period, rather than measuring their BP at home, patients and their spouses were asked to visit the hospital at the usual time of their BP measurement and to bring their own sphygmomanometer. The clinic BP value (CBP) was calculated as the average of the 9 readings taken on these visits by the patients or their spouses. The "white-coat phenomenon" (WCP) was considered to be present when the difference between the CBP and HBP was greater than 20/10 mmHg. The mean reading of home systolic/diastolic BP was 134.7/79.1 mmHg and the mean reading of clinic systolic/diastolic BP was 149.8/86.4 mmHg. In the total subject group, the prevalence rate of WCP was 31%-35% if the WCP was defined as DeltaBP (CBP - HBP) > or =20 mmHg/10 mmHg. In conclusion, ruling out the influence of different factors, including time of day, the sphygmomanometer, the individual taking the BP measurement, the climate, and the patients' health or mood, the WCP was still found to exist to a statistically significant degree. This study indicated that teaching patients to measure their own BP at home is an effective procedure to obtain a more accurate result of their BP level. It also helped to involve the patients more actively in controlling their hypertension.

A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins.

Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6 mg Ig G ml⁻¹ resin and a dissociation constant (K(d)) of 4.78 × 10⁻6 M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.