Clinical Application and Evaluation of Metagenomic Next-Generation Sequencing for Lower Respiratory Tract Infections and Human Tumor Screening.

Background: To evaluate the clinical value of metagenomic next-generation sequencing (mNGS) in screening of lower respiratory tract infections (LRTIs) and human tumors. Methods: Human samples included bronchoalveolar lavage fluid (BALF), sputum, lung biopsy tissue, and peripheral blood from 188 patients who were admitted to our hospital between January 2020 and September 2022 were analyzed using mNGS for simultaneous pathogen and chromosome copy number variation (CNV) detection. Traditional microbial culture and comprehensive microbial test (CMT) were also conducted. The diagnostic efficiencies of the three methods (mNGS, traditional culture, and CMT groups) were compared. Results: Among the 188 patients, 149 (79.3%) were in the LRTIs group and 39 (20.7%) were in the non-LRTIs group. The diagnostic sensitivity and accuracy of the mNGS group were higher than those of the traditional culture and CMT groups (P < 0.001; P < 0.001; P < 0.001; P < 0.001), and the specificity was higher than that of the CMT group (P = 0.039) but lower than that of the traditional culture group (P = 0.006). The positive predictive values of the mNGS and traditional culture groups were higher than that of the CMT group (P = 0.004; P = 0.011). The negative predictive value of the mNGS group was higher than that of the CMT group (P = 0.003). In addition, all samples were subjected to simultaneous chromosome CNV detection, and 8% (15/188) were positive for CNV. Of the 15 patients, 10 were initially misdiagnosed as non-neoplastic diseases, with a misdiagnosis rate of 66.7% (10/15). The BALF CNV test was performed on 13 patients diagnosed with primary or metastatic lung cancer, with a positivity rate of 38.5%. Conclusion: The sensitivity and accuracy of pathogen diagnosis using mNGS were better than those of traditional culture and CMT. CNV detection is an important auxiliary diagnostic tool for cancer, particularly for screening occult tumors.

Adaptive liquid lens with controllable light intensity.

An adaptive liquid lens with controllable light intensity is demonstrated, which can modulate both light intensity and beam spot size. The proposed lens consists of a dyed water solution, a transparent oil, and a transparent water solution. The dyed water solution is used to adjust light intensity distribution by varying the liquid-liquid (L-L) interface. The other two liquids are transparent and designed to control the spot size. In this way, two problems can be solved: the inhomogeneous attenuation of light can be achieved through the dyed layer, and a larger optical power tuning range can be achieved through the two L-L interfaces. Our proposed lens can be used for homogenization effects in laser illumination. In the experiment, an optical power tuning range from - 44.03 m-1 ∼ + 39.42 m-1 and an ∼ 89.84% homogenization level are achieved. Our proposed lens may also ease the vignetting problem in imaging systems.

miR-2188-5p promotes GCRV replication by the targeted degradation of klf2a in Ctenopharyngodon idellus.

Studies on host immunity evasion by aquatic viruses have largely focused on coding genes. There is accumulating evidence for the important biological functions of non-coding miRNAs in virus-host interactions. The regulatory functions of non-coding miRNAs in fish reovirus-host interactions remain unknown. Here, miR-2188-5p in grass carp (Ctenopharyngodon idellus), a miRNA specific to teleosts, was predicted to target the 3' UTR of the transcription factor klf2a. A correlation analysis and dual-luciferase reporter assay revealed that miR-2188-5p could induce the degradation of klf2a. The expression of miR-2188-5p induced the degradation of klf2a in a dose-dependent manner, suppressing the type I interferon response and promoting grass carp reovirus (GCRV) replication. As determined by a co-expression analysis, klf2a inhibited viral infection when miR-2188-5p was overexpressed. The targeted degradation of klf2a by miR-2188-5p could inhibit the type I interferon response and promote the replication of GCRV; however, this targeted degradation ability was insufficient to fully inhibit GCRV infection. These results provide novel insights into the regulatory effects and biological functions of non-coding miRNAs in fish-virus interactions.

Krüppel-like factor 2a (KLF2A) suppresses GCRV replication by upregulating serpinc1 expression in Ctenopharyngodon idellus kidney (CIK) cells.

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Comparison of the Chemical Components, Efficacy and Mechanisms of Action of Chrysanthemum morifolium Flower and Its Wild Relative Chrysanthemum indicum Flower against Liver-Fire Hyperactivity Syndrome of Hypertension via Integrative Analyses.

To clarify the differences in the clinical application scope of Chrysanthemum morifolium flower (CMF) and Chrysanthemum indicum flower (CIF), two herbs of similar origin, an integrated strategy of network pharmacology, molecular pharmacology, and metabolomics was employed, with a view to investigating the commonalities and dissimilarities in chemical components, efficacy and mechanisms of action. Initial HPLC-Q-TOF-MS analysis revealed that CMF and CIF had different flavonoid constituents. The biological processes underlying the therapeutic effects of CMF and CIF on liver-fire hyperactivity syndrome of hypertension (LFHSH) were predicted to be related to inflammatory response, fatty acid production, and other pathways based on network pharmacology analysis. ELISA, molecular docking, Western blot, and metabolomics techniques showed similar effects of CMF and CIF in lowering blood pressure, resistance to tissue, organ and functional damage, and dyslipidemia. However, distinct effects were found in the regulation of inflammatory response, PI3K-Akt and NF-κB signaling pathways, lipid anabolism, renin-angiotensin system, and metabolic abnormalities. The comparable efficacies of CMF and CIF, despite having distinct mechanisms of action, may be attributed to the integration and counteraction of their different regulating capabilities on the above anti-LFHSH mechanisms. This study offers a vital platform for assessment of differential and precise applications of herbs of close origin with similar but slightly different medicinal properties, and provides a research strategy for bridging Chinese medicine and modern precision medicine.

Grass carp SERPINA1 inhibits GCRV infection through degrading CF2.

SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To date, there have been no reports on the immune function of SERPINA1 in fishes. In this study, we first cloned the serpina1 gene of grass carp (Ctenopharyngodon idellus) and found that it could respond rapidly to the infection of Grass carp reovirus (GCRV), and overexpression of serpina1 could enhance the antiviral response of CIK cells. A polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. It was found that SERPINA1 interacted with coagulation factor 2 (CF2) and could degrade it in a dose-dependent manner. In addition, overexpression of cf2 contributed to the infection of GCRV in CIK cells, whereas co-expression of serpina1 and cf2 in grass carp reduced the copy number of GCRV in cells. The results showed that grass carp SERPINA1 could inhibit GCRV infection by degrading CF2. This study proposes that SERPINA1 can inhibit viral infection through interaction with the coagulation factor, providing new insights into the molecular mechanism of SERPINA1's antiviral function.

Screening of Genes Related to Sex Determination and Differentiation in Mandarin Fish (Siniperca chuatsi).

Mandarin fish has an XX/XY sex-determination system. The female mandarin fish is typically larger than the male. Sex identification and the discovery of genes related to sex determination in mandarin fish have important theoretical significance in the elucidation of the regulation and evolutionary mechanism of animal reproductive development. In this study, the chromosome-level genome of a female mandarin fish was assembled, and we found that LG24 of the genome was an X chromosome. A total of 61 genes on the X chromosome showed sex-biased expression. Only six gonadal genes (LG24G00426, LG24G003280, LG24G003300, LG24G003730, LG24G004200, and LG24G004770) were expressed in the testes, and the expression of the other gene LG24G003870 isoform 1 in the ovaries was significantly higher than that in the testes (p < 0.01). Five (except LG24G003280 and LG24G003300) of the seven aforementioned genes were expressed at the embryonic development stage, suggesting their involvement in early sex determination. The expression of LG24G004770 (encoding HS6ST 3-B-like) was also significantly higher in female muscles than in male muscles (p < 0.01), indicating other functions related to female growth. ZP3 encoded by LG24G003870 isoform 1 increased the C-terminal transmembrane domain, compared with that encoded by other fish zp3 isoforms, indicating their different functions in sex determination or differentiation. This study provides a foundation for the identification of sex-determining genes in mandarin fish.

Evaluation of the Effect of Hemolysis on Quantitative Chemiluminescent Immunoassay Results for 10 Analytes.

BACKGROUND: The aim is to evaluate the effect of hemolysis on the quantitative chemiluminescent immunoassay results of 10 analytes and to provide a basis for formulating specific sample rejection criteria and reviewing report results. METHODS: Hemolysis based on the clinical hemolysis index, hemolysis 1+, 2+, and 3+ samples and matched normal samples were collected. The quantitative chemiluminescent immunoassay results of 10 analytes from the two samples (hemolysis and normal) were determined and differences between the results obtained from samples with different degrees of hemolysis and those obtained from normal samples were evaluated. RESULTS: A total of 34 pairs of samples were collected, including 10 pairs of 1+ hemolysis samples, 10 pairs of 2+ hemolysis samples, and 14 pairs of 3+ hemolysis samples. The quantitative chemiluminescence immunoassay detection results for the 10 analytes showed that regardless of the degree of hemolysis, the differences in alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen (CA19-9), luteinizing hormone (LH), folli-cle-stimulating hormone (FSH), and ferritin (FER) between the hemolysis and normal samples were all lower than the total allowable error (TEa) based on biological variation; there were no statistically significant differences between the samples. However, the results for insulin (INS) began to decrease significantly at a hemolytic index of 1+, folic acid (FOL) showed an increase at a hemolytic index of 2+, and there was a significant difference at a he-molytic index of 3+. CONCLUSIONS: This research identified the analytes that are susceptible to hemolysis interference in chemiluminescent immunoassays. The influence of hemolysis on hemolytic clinical laboratory tests was closely related to the assay system used; thus, laboratories should evaluate the effect of hemolysis on their own analysis systems and define assay-specific hemolysis warning indices.

Characterization of SR-B2a and SR-B2b genes and their ability to promote GCRV infection in grass carp (Ctenopharyngodon idellus).

Scavenger receptor class B type 2 (SR-B2) is a pattern recognition receptor involved in innate immunity in mammals; however, the immunological function of SR-Bs in fish remains unclear. In this study, the full-length cDNA sequences of SR-B2a and SR-B2b from grass carp (Ctenopharyngodon idellus) were cloned and designated as CiSR-B2a and CiSR-B2b. Multiple alignments and phylogenetic analyses deduced that CiSR-B2a and CiSR-B2b had the highest evolutionary conservation and were closely related to the zebrafish (Danio rerio) homologs, DrSR-B2a and DrSR-B2b, respectively. Both CiSR-B2a and CiSR-B2b were expressed in all the tested tissues, with the highest expression levels found in the hepatopancreas. In Ctenopharyngodon idellus kidney cells (CIK), CiSR-B2a and CiSR-B2b were mainly located in the cytoplasm, and a small amount located on the plasma membrane. After challenge with Grass Carp Reovirus (GCRV), the expression of CiSR-B2a and CiSR-B2b were significantly upregulated in the spleen (about 10.27 and 27.19 times higher than that at 0 day, p < 0.01). With CiSR-B2a or CiSR-B2b overexpressed in CIK, the relative copy number of GCRV in the cells was both significantly increased compared to that in the control group, indicating that CiSR-B2a and CiSR-B2b may be important proteins during the infection processes of GCRV.

Genome-wide identification, evolution of Krüppel-like factors (klfs) and their expressions during GCRV challenge in grass carp (Ctenopharyngodonidella).

The Krüppel-like factors (KLFs) are a family of transcription factors containing three highly conserved tandem zinc finger structures, and each member participates in multiple physiological and pathological processes. The publication of genome sequences and the application of bioinformatics tools have led to the discovery of numerous gene families in fishes. Here, 24 klf genes were re-annotated in grass carp. Subsequently, the number of klf family members were investigated in some representative vertebrate species. Then, a series of bioinformatics analysis showed that grass carp klfs in the same subfamily had similar genome structure patterns and conserved distribution patterns of motifs, which supported their molecular evolutionary relationships. Furthermore, the mRNA expression profiles showed that 24 grass carp klfs were ubiquitously expressed in 11 different tissues, and some of them displayed tissue-enriched expression patterns. Finally, the expressions of the evolutionarily expanded klf members (klf2a, 2b, 2l, 5a, 5b, 5l, 6a, 6b, 7a, 7b, 11a, 11b, 12a, 12b, 15 and 15l) during GCRV infection were also analyzed. The results suggested that grass carp klf genes with common evolutionary sources may share functional diversity and conservation. In conclusion, this study provides preliminary clues for further researches on grass carp klf members and their underlying transcriptional regulatory mechanisms during GCRV infection.

Diagnostic value of urinary microprotein concentration for patients with negative urinary protein test results and positive urinary casts on microscopic examination.

OBJECTIVE: To analyze the association between positive urinary casts on microscopic examination and urinary microprotein concentration in the case of negative urinary protein test results. This study also investigated the diagnostic value of urinary microprotein examination. SUBJECTS: A total of 949 samples that were analyzed with a UF-1000i Urine Analyzer and returned cast alarm results were categorized into two groups, a positive and negative group, according to qualitative urinary protein sulfosalicylic acid test results. Then, 54 samples with negative protein test results but positive cast results according to microscopic examination were selected as the study group; 60 normal people with healthy physical examination results were selected as the control group. Both groups underwent urinary microprotein tests, including urinary microalbumin (mAlb), α1-microglobulin (A1M), transferrin (TRU), and immunoglobulin G (IgG). T tests were used to evaluate mean differences between groups and chi-square tests were used to calculate ratio differences between groups. RESULTS: (a) Microscopic examinations of the positive and negative protein groups revealed no statistically significant difference in cast detection rate (P = .421). (b) Among the 54 samples in the study group, 37 were found to have abnormal casts, while in the remaining 17 samples, only hyaline casts were detected. (c) The detection levels of mAlb, A1M, and IgG in the study group were significantly higher than the control group (P values < .05). CONCLUSION: Urinary microprotein test should be included in the re-examination rules for routine tests for patients with negative protein results and positive casts under microscopic examination.

Qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from Senecio scandens to Senecio scandens-containing preparations by high performance liquid chromatography-tandem mass spectrometry.

Senecio scandens as a commonly used traditional Chinese medicine that is used alone or in combination with other herbs in preparations such as QianBai BiYan tablets has attracted much attention because of its hepatotoxic pyrrolizidine alkaloids. Nowadays, most studies for pyrrolizidine alkaloids are only performed on herbs or a preparation, however, production of preparations is a dynamic process, control of toxic impurities for raw materials, or finished products cannot monitor the production process dynamically. Thus, in this study, qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from S. scandens to its preparations was carried out with HPLC-MS/MS for the first time, which was more comprehensive and dynamic than the previous single-layer analysis. First, the species of pyrrolizidine alkaloids in S. scandens were analyzed, and the characteristic fragmentation rules of pyrrolizidine alkaloids containing common parent nucleus were found, which can be used to identify these components rapidly in the future. Then, a quantitative method for S. scandens to QianBai BiYan tablets and other nine S. scandens-containing preparations was established, and after the medication safety speculation, all of them met the relevant safety requirements. After that, in order to ensure the stability and controllable of drug quality, the limit of pyrrolizidine alkaloids in preparations was determined according to the safe dosage that is stipulated to be the same as raw materials. Finally, the factors causing the content change of pyrrolizidine alkaloids in S. scandens from different source were studies, which can provide theoretical basis for selecting suitable raw materials for production.

Characterization and expression of galectin-3 in grass carp (Ctenopharyngodon idella).

Galectins are members of evolutionary conserved lectin family and play important roles in the innate and adaptive immunity of both vertebrates and invertebrates. Galectin-3 is the only chimera galectin with one C-terminal carbohydrate recognition domain (CRD) connected to the N-terminal end. Here, a galectin-3 (named CiGal3) from grass carp was identified and characterized, which encodes polypeptides 362 amino acids with a predicted molecular mass of 36.45 kDa and theoretical isoelectric point of 4.91. The sugar binding motifs involved in carbohydrate binding activity (H-N-R, V-N and W--E-R) were detected in CRD. In comparison to other species, CiGal3 showed the highest similarity and identity to Cyprinus carpio (95.3% sequence similarity and 92.5% sequence identity). The subcellular localization of CiGal3 was distributed in the cytoplasm and nucleus of transfected cells. The CiGal3 transcripts were ubiquitously expressed in all checked tissues and highly expressed in immune tissues. In addition, the expression of CiGal3 in liver and spleen was induced post grass carp reovirus (GCRV), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C) challenge. These results suggest that CiGal3 plays a vital role in the immune system.

Characterisation and function of TRIM23 in grass carp (Ctenopharyngodon idella).

Tripartite motif (TRIM) proteins are key components of the innate immune system, functioning as antiviral restriction factors or modulating signaling cascades that lead to proinflammatory cytokine induction. In the present study, the TRIM family gene TRIM23 from grass carp (Ctenopharyngodon idella) was cloned and characterised. TRIM23 was moderately expressed in the examined tissues, and the significantly altered expression was observed after grass carp reovirus (GCRV) and poly(I:C) infection. Dual-luciferase activity assay showed that TRIM23, especially its C-terminal domain ARF, depressed the promoter activity of IRF3 and IRF7. The subcellular localisation showed that TRIM23 protein was located in the cytoplasm and could be recruited by both TRAF6 and MyD88. Furthermore, TRIM23 was confirmed to interact with either TRAF6 or MyD88 by the bimolecular fluorescence complementation (BiFC) system in CIK cells. Additionally, autophagy was enhanced by over-expressed TRIM23 in 293T cells. Taken together, our results demonstrate that TRIM23 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the TRIM23 in teleosts.

[Optical Design of Miniature Infrared Gratings Spectrometer Based on Planar Waveguide].

In order to miniaturize an infrared spectrometer, we analyze the current optical design of miniature spectrometers and propose a method for designing a miniature infrared gratings spectrometer based on planar waveguide. Common miniature spectrometer uses miniature optical elements to reduce the size of system, which also shrinks the effective aperture. So the performance of spectrometer has dropped. Miniaturization principle of planar waveguide spectrometer is different from the principle of common miniature spectrometer. In planar waveguide spectrometer, the propagation of light is limited in a thin planar waveguide, which looks like the whole optical system is squashed flat. In the direction parallel to the planar waveguide, the light through the slit is collimated, dispersed and focused. And a spectral image is formed in the detector plane. This propagation of light is similar to the light in common miniature spectrometer. In the direction perpendicular to the planar waveguide, light is multiple reflected by the upper and lower surfaces of the planar waveguide and propagates in the waveguide. So the size of corresponding optical element could be very small in the vertical direction, which can reduce the size of the optical system. And the performance of the spectrometer is still good. The design method of the planar waveguide spectrometer can be separated into two parts, Czerny-Turner structure design and planar waveguide structure design. First, by using aberration theory an aberration-corrected (spherical aberration, coma, focal curve) Czerny-Turner structure is obtained. The operation wavelength range and spectral resolution are also fixed. Then, by using geometrical optics theory a planar waveguide structure is designed for reducing the system size and correcting the astigmatism. The planar waveguide structure includes a planar waveguide and two cylindrical lenses. Finally, they are modeled together in optical design software and are optimized as a whole. An infrared planar waveguide spectrometer is designed using this method. The operation wavelength range is 8 - 12 µm, the numerical aperture is 0.22, and the linear array detector contains 64 elements. By using Zemax software, the design is optimized and analyzed. The results indicate that the size of the optical system is 130 mm x 125 mm x 20 mm and the spectral resolution of spectrometer is 80 nm, which satisfy the requirements of design index. Thus it is this method that can be used for designing a miniature spectrometer without movable parts and sizes in the range of several cubic centimeters.