Eyeless razor clam Sinonovacula constricta discriminates light spectra through opsins to guide Ca2+ and cAMP signaling pathways.

Phototransduction is based on opsins that drive distinct types of Gα cascades. Although nonvisual photosensitivity has long been known in marine bivalves, the underlying molecular basis and phototransduction mechanism are poorly understood. Here, we introduced the eyeless razor clam Sinonovacula constricta as a model to clarify this issue. First, we showed that S. constricta was highly diverse in opsin family members, with a significant expansion in xenopsins. Second, the expression of putative S. constricta opsins was highly temporal-spatio specific, indicating their potential roles in S. constricta development and its peripheral photosensitivity. Third, by cloning four S. constricta opsins with relatively higher expression (Sc_opsin1, 5, 7, and 12), we found that they exhibited different expression levels in response to different light environments. Moreover, we demonstrated that these opsins (excluding Sc_opsin7) couple with Gαq and Gαi cascades to mediate the light-dependent Ca2+ (Sc_opsin1 and 5) and cAMP (Sc_opsin12) signaling pathways. The results indicated that Sc_opsin1 and 5 belonged to Gq-opsins, Sc_opsin12 belonged to Gi-opsins, while Sc_opsin7 might act as a photo-isomerase. Furthermore, we found that the phototransduction function of S. constricta Gq-opsins was dependent on the lysine at the seventh transmembrane domain, and greatly influenced by the external light spectra in a complementary way. Thus, a synergistic photosensitive system mediated by opsins might exist in S. constricta to rapidly respond to the transient or subtle changes of the external light environment. Collectively, our findings provide valuable insights into the evolution of opsins in marine bivalves and their potential functions in nonvisual photosensitivity.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A.

BACKGROUND: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. METHODS: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. RESULTS: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. CONCLUSIONS: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Effectiveness of a socioecological model-guided, smart device-based, self-management-oriented lifestyle intervention in community residents: protocol for a cluster-randomized controlled trial.

BACKGROUND: Healthy lifestyles are crucial for preventing chronic diseases. Nonetheless, approximately 90% of Chinese community residents regularly engage in at least one unhealthy lifestyle. Mobile smart devices-based health interventions (mHealth) that incorporate theoretical frameworks regarding behavioral change in interaction with the environment may provide an appealing and cost-effective approach for promoting sustainable adaptations of healthier lifestyles. We designed a randomized controlled trial (RCT) to evaluate the effectiveness of a socioecological model-guided, smart device-based, and self-management-oriented lifestyles (3SLIFE) intervention, to promote healthy lifestyles among Chinese community residents. METHODS: This two-arm, parallel, cluster-RCT with a 6-month intervention and 6-month follow-up period foresees to randomize a total of 20 communities/villages from 4 townships in a 1:1 ratio to either intervention or control. Within these communities, a total of at least 256 community residents will be enrolled. The experimental group will receive a multi-level intervention based on the socioecological model supplemented with a multi-dimensional empowerment approach. The control group will receive information only. The primary outcome is the reduction of modifiable unhealthy lifestyles at six months, including smoking, excess alcohol consumption, physical inactivity, unbalanced diet, and overweight/obesity. A reduction by one unhealthy behavior measured with the Healthy Lifestyle Index Score (HLIS) will be considered favorable. Secondary outcomes include reduction of specific unhealthy lifestyles at 3 months, 9 months, and 12 months, and mental health outcomes such as depression measured with PHQ-9, social outcomes such as social support measured with the modified Multidimensional Scale of Perceived Social Support, clinical outcomes such as obesity, and biomedical outcomes such as the development of gut microbiota. Data will be analyzed with mixed effects generalized linear models with family and link function determined by outcome distribution and accounting for clustering of participants in communities. DISCUSSION: This study will provide evidence concerning the effect of a mHealth intervention that incorporates a behavioral change theoretical framework on cultivating and maintaining healthy lifestyles in community residents. The study will provide insights into research on and application of similar mHealth intervention strategies to promote healthy lifestyles in community populations and settings. TRIAL REGISTRATION NUMBER: ChiCTR2300070575. Date of registration: April 17, 2023. https://www.chictr.org.cn/index.aspx .

Quantitative Magnetic Resonance Imaging Had Greater Sensitivity in Diagnosing Chondral Lesions of the Knee: A Systematic Review and Meta-Analysis.

PURPOSE: To investigate the accuracy and reliability of magnetic resonance imaging (MRI) in identifying and grading chondral lesions and explore the optimal imaging technique to image cartilage. METHOD: A comprehensive search was conducted on Medline, Embase, and Cochrane Library. Eligible cohort studies published before August 2022 were included. The study reports used MRI to diagnose and grade cartilage lesions, with intraoperative findings as the reference standard. Summary estimates of diagnostic performance were obtained. The reliability of MRI interpretation was summarized. Subgroup analyses were performed based on assessed imaging techniques, field strength, and joint surface. RESULTS: Forty-three trials and 3,706 patients were included in the systematic review. The overall area under curve for hierarchical summarized receiver operating characteristics was 0.91 (95% confidence interval [CI] 0.88-0.93). The pooled sensitivity for quantitative MRI, 3-dimensional MRI, and 2-dimensional MRI was 0.82 (95% CI 0.64-0.92), 0.79 (95% CI 0.74-0.83), and 0.63 (95% CI 0.51-0.73), respectively. The pooled sensitivity of 3 Tesla (3T), 1.5 Tesla (1.5T), and <1.5 Tesla MRI was 0.79 (95% CI 0.72-0.85), 0.67 (95% CI 0.60-0.74), and 0.55 (95% CI 0.39-0.71), respectively. There were differences in interobserver consistency across different studies. CONCLUSIONS: In general, MRI had high specificity in discriminating normal cartilage, but its sensitivity for identifying chondral lesions is less optimal. Further analysis showed that quantitative MRI, 3D MRI, and 3T MRI demonstrate greater sensitivity compared with 2D MRI, 1.5T MRI, and <1.5 Tesla MRI. LEVEL OF EVIDENCE: Level III, systematic review of Level II-III studies.

First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand.

OBJECTIVES: Translocator protein 18 kDa (TSPO) positron emission tomography (PET) can be harnessed for the non-invasive detection of macrophage-driven inflammation. [18F]LW223, a newly reported TSPO PET tracer which was insensitive to rs6971 polymorphism, showed favorable performance characteristics in a recent imaging study involving a rat myocardial infarction model. To enable quantitative neuroimaging with [18F]LW223, we conducted kinetic analysis in the non-human primate (NHP) brain. Further, we sought to assess the utility of [18F]LW223-based TSPO imaging in a first-in-human study. METHODS: Radiosynthesis of [18F]LW223 was accomplished on an automated module, whereas molar activities, stability in formulation, lipophilicity and unbound free fraction (fu) of the probe were measured. Brain penetration and target specificity of [18F]LW223 in NHPs were corroborated by PET-MR imaging under baseline and pre-blocking conditions using the validated TSPO inhibitor, (R)-PK11195, at doses ranging from 5 to 10 mg/kg. Kinetic modeling was performed using one-tissue compartment model (1TCM), two-tissue compartment model (2TCM) and Logan graphical analyses, using dynamic PET data acquisition, arterial blood collection and metabolic stability testing. Clinical PET scans were performed in two healthy volunteers (HVs). Regional brain standard uptake value ratio (SUVr) was assessed for different time intervals. RESULTS: [18F]LW223 was synthesized in non-decay corrected radiochemical yields (n.d.c. RCYs) of 33.3 ± 6.5% with molar activities ranging from 1.8 ± 0.7 Ci/µmol (n = 11). [18F]LW223 was stable in formulation for up to 4 h and LogD7.4 of 2.31 ± 0.13 (n = 6) and fu of 5.80 ± 1.42% (n = 6) were determined. [18F]LW223 exhibited good brain penetration in NHPs, with a peak SUV value of ca. 1.79 in the whole brain. Pre-treatment with (R)-PK11195 substantially accelerated the washout and attenuated the area under the time-activity curve, indicating in vivo specificity of [18F]LW223 towards TSPO. Kinetic modeling demonstrated that 2TCM was the most suitable model for [18F]LW223-based neuroimaging. Global transfer rate constants (K1) and total volumes of distribution (VT) were found to be 0.10 ± 0.01 mL/cm3/min and 2.30 ± 0.17 mL/cm3, respectively. Dynamic PET data analyses across distinct time windows revealed that the VT values were relatively stable after 60 min post-injection. In a preliminary clinical study with two healthy volunteers, [18F]LW223 exhibited good brain uptake and considerable tracer retention across all analyzed brain regions. Of note, an excellent correlation between SUVr with VT was obtained when assessing the time interval from 20 to 40 min post tracer injection (SUVr(20-40 min), R2 = 0.94, p < 0.0001), suggesting this time window may be suitable to estimate specific binding to TSPO in human brain. CONCLUSION: Our findings indicate that [18F]LW223 is suitable for quantitative TSPO-targeted PET imaging in higher species. Employing state-of-the-art kinetic modeling, we found that [18F]LW223 was effective in mapping TSPO throughout the NHP brain, with best model fits obtained from 2TCM and Logan graphical analyses. Overall, our results indicate that [18F]LW223 exhibits favorable tracer performance characteristics in higher species, and this novel imaging tool may hold promise to provide effective neuroinflammation imaging in patients with neurological disease.

Mutations in DNA binding domain of p53 impede RSL1D1-p53 interaction to escape from degradation in human colorectal cancer cells.

Different from the nucleolus-specific localization in some types of cancer cells, ribosomal L1 domain-containing protein 1 (RSL1D1) distributes throughout the nucleus in human colorectal cancer (CRC) cells. RSL1D1 directly interacts with DNA binding domain (aa 93-292) of wild-type p53 (p53-WT) and thereby recruits p53 to HDM2. The ensuing formation of RSL1D1/HDM2/p53 complex enhances p53 ubiquitination and decreases the protein level of p53 in CRC cells. In this study, we investigated the interaction between RSL1D1 and mutant p53 proteins. We first corroborated that aa 93-224 of p53 is a more precise domain for RSL1D1 binding and mutation in either aa 93-224 or aa 225-292 domain of p53 affects RSL1D1-p53 interaction. R175H mutated p53 does not interact with RSL1D1, whereas R273H mutated p53 still can bind to RSL1D1 but showing a remarkably decreased affinity than p53-WT. Although p53-R273H retains a weakened binding affinity with RSL1D1, it can hardly be recruited to HDM2 by RSL1D1 in HCT116 CRC cells. Accordingly, RSL1D1 loses its capacity to negatively regulate either R175H or R273H p53 mutant via directly interaction in HCT116 cells, thereby facilitating p53 mutants to accumulate and gain oncogenic function. Our findings help explain why mutant p53 proteins are more stable than p53-WT in CRC cells.

Feasibility of using software-aided selection of virtual monoenergetic level for optimal image quality of acute necrotising pancreatitis based on dual-energy computed tomography: a preliminary study.

OBJECTIVE: This study aimed to assess the feasibility of software-aided selection of monoenergetic level for acute necrotising pancreatitis (ANP) depiction compared to other automatic image series generated using dual-energy computed tomography (CT). METHODS: The contrast-enhanced dual-source dual-energy CT images in the portal venous phase of 48 patients with ANP were retrospectively analysed. Contrast-to-noise ratio (CNR) of pancreatic parenchyma-to-necrosis, signal-to-noise ratio (SNR) of the pancreas, image noise, and score of subjective diagnosis were measured, calculated, and compared among the CT images of 100 kV, Sn140 kV, weighted-average 120 kV, and optimal single-energy level for CNR. RESULTS: CNR of pancreatic parenchyma-to-necrosis in the images of 100 kV, Sn140 kV, weighted-average 120 kV, and the optimal single-energy level for CNR was 5.18 ± 2.39, 3.13 ± 1.35, 5.69 ± 2.35, and 9.99 ± 5.86, respectively; SNR of the pancreas in each group was 6.31 ± 2.77, 4.27 ± 1.56, 7.21 ± 2.69, and 11.83 ± 6.30, respectively; image noise in each group was 18.78 ± 5.20, 17.79 ± 4.63, 13.28 ± 3.13, and 9.31 ± 2.96, respectively; and score of subjective diagnosis in each group was 3.56 ± 0.50, 3.00 ± 0.55, 3.48 ± 0.55, and 3.88 ± 0.33, respectively. The four measurements of the optimal single-energy level for CNR images were significantly different from those of images in the other three groups (P < 0.05). CNR of pancreatic parenchyma-to-necrosis, SNR of the pancreas, and score of subjective diagnosis in the images of the optimal single-energy level for CNR were significantly higher, while the image noise was lower than those in the other three groups (all P = 0.000). CONCLUSION: Optimal single-energy level imaging for CNR of dual-source CT could improve quality of CT images in patients with ANP, enhancing the display of necrosis in the pancreas.

Impact of 24-Hour Intraocular Pressure on Optic Nerve Fiber Layer Thickness in Patients with Early Diabetic Retinopathy.

Background: The optic nerve fiber layer, composed of ganglion cell axons within the ganglion cell layer, undergoes thickness changes due to diabetic retinopathy. However, the relationship between intraocular pressure (IOP) and optic fiber layer thickness remains unclear. Objective: To investigate the correlation between 24-hour intraocular pressure and optic nerve fiber layer thickness in patients with early diabetic retinopathy. Methods: This retrospective study collected 353 patients with early diabetic retinopathy from January 2019 to December 2021. They were categorized into the retinopathy group (n = 153) and the control group (n = 200). 24-hour IOP and optic fiber layer thickness were assessed, and the correlation between them was analyzed. Results: The observation group exhibited significantly higher 24-hour IOP compared to the control group (16.64 ± 2.58 vs. 15.63 ± 2.52 mmHg, P < .001). Notably, the thickness of upper, lower, nasal, temporal, and average optic nerve fiber layers in the observation group decreased significantly (P < .001). Pearson linear correlation revealed significant negative associations between 24-hour IOP and upper, nasal, temporal, and mean optic nerve fiber layer thickness (R2 = -0.277, -0.399, -0.344, and -0.489, P < .05). The upper, lower, nasal, temporal, and mean optic fiber thickness demonstrated diagnostic value for non-early diabetic retinopathy in type 2 diabetes patients (P < .05), with mean optic fiber thickness displaying the highest diagnostic potential (area under the curve: 0.843, 95% Confidence Interval: 0.803-0.884, P < .001). Conclusions: Thinning of the optic nerve fiber layer in early diabetic retinopathy patients holds predictive value for the condition and exhibits a negative correlation with 24-hour intraocular pressure.

First report of Nigrospora musae causing white leaf spot of Basella alba L in China.

Basella alba L, an edible annual twining herb of the genus Basella and the family Basella, has been widely introduced and cultivated in China. Basella alba L. as a leaf vegetable, is rich in vitamins A and C, iron, and calcium (FAO 1988). In May 2022, severe white leaf spots were observed in plantation located in Shuangfeng County (27°41'36" N, 111°56'60" E), Hunan Province, China. More than 50 Basella alba L plants were surveyed with over 80% disease incidence in an area of 300 square meters of greenhouse. The symptoms on leaves were initially small purplish-brown lesions from leaf margins or tips, with lesions expanded, the middle of the lesions was yellowish-white to yellowish-brown, slightly dented. The edge of lesions was purplish-brown, with obvious boundary between the diseased parts and the non-diseased ones. A total of 20 symptomatic samples were randomly collected. Lesion margins were surface sterilized in 2% sodium hypochlorite for 1 min, rinsed with sterile distilled water for three times, dried, placed on potato dextrose agar (PDA), and incubated at 25°C and 60% relative humidity in the dark for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. Six isolates were obtained. Colonies were fast-growing, massive sparse aerial hyphae, initially white, turning gray and black after 7 days. Hyphae were branched, septa, and transparent. To induce sporulation, colonies were transferred to sodium carboxymethyl cellulose (CMC) plates (Z. M. Wen., & X. Y. Luo 1991). Conidia were single-celled, dark black, oblate, or nearly spherical, and measured 10.2 to 15.1 µm × 9.7 to 16.0 µm in diameter (n=50). For molecular identification, the rDNA internal transcribed spacer (ITS), the ß-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified from genomic DNA by primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass & Donaldson. 1995), and EF1-728F/EF1-986R (Carbone & Kohn, 1999). The sequences of six isolates (L1, L2, L7, L10, L11, L12) were deposited in GenBank with accession numbers OP703335, OP703336, OP703337, OP703338, OP703339, OP703340 (ITS), OP784252, OP784157, OP784253, OP784254, OP784255, OP784256 (TEF-1α), and OP724156, OP724158, OP779771, OP779772, OP779773, OP779774 (TUB2). A blast search of sequences showed the ITS, TEF-1α, and TUB2 sequences had >98% identity with homologue sequences from Nigrospora musae isolates BRJ2 (OP451019.1), CBS 319.34 (KY019419.1) and LC6385 (KY019567.1), respectively. These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as Nigrospora musae (Wang, 2017). Pathogenicity test was carried out in plants. Strains were cultured on CMC plates for 14 days, then the mycelium was scraped to make conidial suspension (1×106 conidia/mL). After 5-6 leaves of the Basella alba L were sprouted, conidial suspension was sprayed directly on the leaves, with leaves sprayed by sterile distilled water as the control. All plants were kept in the greenhouse with temperature at 25/30°C (night/day) and 75% relative humidity. After 7 days, symptoms were observed on inoculated leaves of plants, which were the same as previously described samples, while the control plants showed no symptoms. The test was repeated three times with similar results. The strains reisolated from the inoculated leaves were morphologically identical to Nigrospora musae, conforming to Koch's postulates. symptoms of Nigrospora musae is similar to that of the other leaf diseases of Basella alba L reported in China. (H. P. Jiang.2000; S. Tan.1996). To our knowledge, this is the first report of Nigrospora musae causing white leaf spot of Basella alba L in China. The pathogen may severely threat the production of Basella alba L. The information on identification of this fungus may be helpful to the control and prevention of the disease. References: 1. FAO. 1988. Page 103 in: Traditional Food Plants: A Resource Book for Promoting the Exploitation and Consumption of Food Plants in Arid, Semi-arid and Sub-humid Lands of Eastern Africa. FAO Food and Nutrition Paper 42. FAO, Rome, Italy. 2. Z. M. Wen., & X. Y. Luo. Fusarium graminearum spore production medium filtering [J]. Chinese journal of food hygiene, 1991 (04): 11-13. 3. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. 4. Carbone, I., et al. 1999. Mycologia. 91: 553-556. 5. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61: 1323. 6. Wang, 2017. Phylogenetic reassessment of Nigrospora: Ubiquitous endophytes, plant, and human pathogens. 7. H. P. Jiang., et al. Occurrence and comprehensive control of white leaf spot of Basella alba L [J]. Plant Protection Technology and Extension, 2000(02):19. 8. S. Tan. The symptoms and control measures of white leaf spot of Basella alba L [J]. Anhui Agricultural, 1996(08):15. *Indicates the corresponding author. Kaifa Guo, E-mail: andygkf@126.com.

Optical detection using CRISPR-Cas12a of Helicobacter pylori for veterinary applications.

Helicobacter pylori (H. pylori) is a zoonotic gastric microorganism capable of efficient interspecies transmission. Domesticated companion animals, particularly dogs and cats, serve as natural reservoirs for H. pylori. This phenomenon facilitates the extensive dissemination of H. pylori among households with pets. Hence, the prompt and precise identification of H. pylori in companion animals holds paramount importance for the well-being of both animals and their owners. With the assistance of Multienzyme Isothermal Rapid Amplification (MIRA) and CRISPR-Cas12a system, we successfully crafted a highly adaptable optical detection platform for H. pylori. Three sensor systems with corresponding visual interpretations were proposed. This study demonstrated a rapid turnaround time of approximately 45 min from DNA extraction to the result display. Moreover, this platform topped germiculture and real-time PCR in terms of sensitivity or efficiency in clinical diagnoses of 66 samples. This platform possesses significant potential as a versatile approach and represents the premiere application of CRISPR for the non-invasive detection of H. pylori in companion animals, thereby mitigating the dissemination of H. pylori among household members.

The Loss-Function of KNL1 Causes Oligospermia and Asthenospermia in Mice by Affecting the Assembly and Separation of the Spindle through Flow Cytometry and Immunofluorescence.

KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence.

[Exploring the mechanisms of Jujing pill on varicocele-associated male infertility via network pharmacology and molecular docking technology].

OBJECTIVE: To investigate the potential mechanism of treating varicocele-associated male infertility with Jujing pill using network pharmacology and molecular docking technology. METHODS: The TCMSP and BATMAN databases were used to search for the Chinese medicine components of the Jujing pill and obtain the corresponding targets. The databases GeneCards, DISGENET, OMIM, and HPO were searched for 'varicocele' and 'male infertility' to identify the potential targets of varicocele-associated male infertility. Wayne diagrams were drawn using the jvenn tool to determine the intersection targets of the Chinese medicine targets and disease targets. The intersecting targets were further analyzed to identify the components and Chinese medicine corresponding to them. A Chinese medicine-active ingredient-target network map was constructed in Cytoscape 3.8.2. The protein-protein interaction (PPI) network of the intersecting targets was constructed using the STRING platform. The intersecting targets were imported into the DAVID database for GO enrichment analysis and KEGG-based pathway enrichment analysis. The KEGG database was used to select the most relevant pathway to the topic, and a KEGG pathway map was constructed using the mapper tool. The top 15 pathways with FDR values and their related targets and components were used to draw a core ingredient-target-pathway map. Finally, molecular docking was performed to verify the protein receptors and small molecule ligands of the core genes, and the results were visualized using AutoDock and PyMol software. RESULTS: A total of 207 ingredients and 1103 predicted targets of Jujing pill were screened. Additionally, 285 targets of varicocele were also identified. By using a Venn diagram, 86 common targets were obtained. The analysis of Gene Ontology (GO) results revealed significant enrichment in various biological processes (BP) such as positive regulation of gene expression, positive regulation of transcription, positive and negative regulation of apoptotic processes, response to hypoxia, response to estradiol, and positive regulation of nitric oxide biosynthesis processes. Furthermore, significant enrichment in cellular components (CC) was observed in macromolecules, cytoplasm, nucleus, and phosphatidylinositol 3-kinase complex. In terms of molecular function (MF), enrichment was found in enzyme binding, identical protein binding, transcriptional co-activator binding, and others. KEGG analysis demonstrated enrichment in pathways related to cancer, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, FoxO signaling pathway, and more. Molecular docking results indicated that the core ingredients exhibited a strong binding capacity with the key targets. Conclusion： The effective active ingredients of Jujing pill exert their therapeutic effects on varicocele-associated male infertility through multiple targets and pathways. These findings provide a theoretical basis for future cell and animal experiments to verify the mechanism of action of Jujing pill in treating varicocele-associated male infertility.

[Observation of effect of Wu Yun Liu Qi on reproductive hormones in varicocele patients].

Objective： To observe roproductive hormone levels in varicocele patients during a cycle (6 years) of Wu Yun Liu Qi, and explore whether the cycle had effect on the roproductive hormone levels. Methods： Data of roproductive hormone levels in varicocele patients from 2015 to 2020 were analyzed retrospectively. FSH、LH、T、PRL、E2 levels and T/E2 ratio were compared among the six years. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year from 2015 to 2020 its yunqi characteristics were determined. Results: Totally data of 848 cases of varicocele patients were collected from 2015 to 2020. Among which, in 2015 (Yiwei year) there were 57 cases, in 2016 (Bingshen year) 83 cases, in 2017 (Dingyou year ) 133 cases, in 2018(Wuxu year) 156 cases, in 2019(Sihai year) 274 cases, and in 2020(Gengzi year) 145 cases. The levels of FSH、LH、PRL、T were not diferrent statistically from the six years except individual year. However, the level of E2 in 2016 when the Yunqi was Shao Yang Xiang Huo Si Tian and Jue Yin Feng Mu Zai Quan obviously higher than other years excpet 2018( All P< 0.05). And T/E2 ratio was lower in 2016 than other years except 2018 and 2020( All P< 0.05). Conclusions: Shi Xiang factors of Wu Yun Liu Qi had effect on roproductive hormone levels in varicocele patients, showing by higher E2 level in Yinshen year when the Shi Xiang factors may have bad effect on human fertility.

Suppression of EZH2 inhibits TGF-ß1-induced EMT in human retinal pigment epithelial cells.

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is critically involved in the occurrence of subretinal fibrosis. This study aimed to investigate the role of enhancer of zeste homolog 2 (EZH2) in EMT of human primary RPE cells and the underlying mechanisms of the anti-fibrotic effect of EZH2 suppression. Primary cultures of human RPE cells were treated with TGF-ß1 for EMT induction. EZH2 was silenced by siRNA to assess the expression levels of epithelial and fibrotic markers using qRT-PCR, Western blot, and immunofluorescence staining assay. Furthermore, the cellular migration, proliferation and barrier function of RPE cells were evaluated. RNA-sequencing analyses were performed to investigate the underlying mechanisms of EZH2 inhibition. Herein, EZH2 silencing up-regulated epithelial marker ZO-1 and downregulated fibrotic ones including α-SMA, fibronectin, and collagen 1, alleviating EMT induced by TGF-ß1 in RPE cells. Moreover, silencing EZH2 inhibited cellular migration and proliferation, but didn't affect cell apoptosis. Additionally, EZH2 suppression contributed to improved barrier functions after TGF-ß1 stimulation. The results from RNA sequencing suggested that the anti-fibrotic effect of EZH2 inhibition was associated with the MAPK signaling pathway, cytokine-cytokine receptor interaction, and the TGF-beta signaling pathway. Our findings provide evidence that the suppression of EZH2 might reverse EMT and maintain the functions of RPE cells. EZH2 could be a potential therapeutic avenue for subretinal fibrosis.

Pathogenicity comparison between QX-type and Mass-type infectious bronchitis virus to different segments of the oviducts in laying phase.

BACKGROUND: The QX-type infectious bronchitis virus (IBV) has become the predominant genotype worldwide in recent years and has caused serious economic losses to the chicken industry. The most significant feature of QX IBV is that its infection in the early growing stage can cause abnormal oviduct development, resulting in a high proportion of 'false layers' in poultry flocks of laying hens and breeders. However, few studies have evaluated whether infections of QX-type IBV in laying stages can also cause severe pathological changes in the oviduct. METHODS: In this study, 300-day-old specific-pathogen-free chickens were infected either with the QX-type strain QXL or Massachusetts (Mass)-type strain M41 to compare their pathogenicity on different segments of the oviduct. RESULTS: Both the QXL and M41 strains successfully replicated in all segments of the oviduct; however, the QXL strain was more highly distributed in mucosal layer and caused severe lesions in the lamina propria, including interstitial dilation, inflammatory cell infiltration, and distinct expansion of tubular glands. Moreover, the QXL strain induced high expression of proinflammatory cytokines and cytotoxic molecules in the majority of segments in the oviduct. Further research found that the QXL strain may affected the formation of shell membranes and eggshells by inhibiting the expression of type I collagen and CaBP-D28k. CONCLUSIONS: Our results indicate that the QX-type IBV is more pathogenic than Mass-type IBV to oviduct in laying phase. Collectively, these findings provide detailed information on the pathological changes in different segments of the oviduct in laying phase, which could offer a better understanding about the pathogenicity of IBV.

High-strength deep learning image reconstruction in coronary CT angiography at 70-kVp tube voltage significantly improves image quality and reduces both radiation and contrast doses.

OBJECTIVES: To explore the use of 70-kVp tube voltage combined with high-strength deep learning image reconstruction (DLIR-H) in reducing radiation and contrast doses in coronary CT angiography (CCTA) in patients with body mass index (BMI) < 26 kg/m2, in comparison with the conventional scan protocol using 120 kVp and adaptive statistical iterative reconstruction (ASIR-V). METHODS: A total of 100 patients referred to CCTA were prospectively enrolled and randomly divided into two groups: low-dose group (n = 50) with 70 kVp, Smart mA for noise index (NI) of 36HU, contrast dose rate of 16mgI/kg/s, and DLIR-H, and conventional group (n = 50) with 120 kV, Smart mA for NI of 25HU, contrast dose rate of 32mgI/kg/s, and 60%ASIR-V. Radiation and contrast dose, subjective image quality score, and objective image quality measurement (image noise, contrast-noise-ratio (CNR), and signal-noise-ratio (SNR) for vessel) were compared between the two groups. RESULTS: Low-dose group used significantly reduced contrast dose (23.82 ± 3.69 mL, 50.6% reduction) and radiation dose (0.75 ± 0.14 mSv, 54.5% reduction) compared to the conventional group (48.23 ± 6.38 mL and 1.65 ± 0.66 mSv, respectively) (all p < 0.001). Both groups had similar enhancement in vessels. However, the low-dose group had lower background noise (23.57 ± 4.74 HU vs. 35.04 ± 8.41 HU), higher CNR in RCA (48.63 ± 10.76 vs. 29.32 ± 5.52), LAD (47.33 ± 10.20 vs. 29.27 ± 5.12), and LCX (46.74 ± 9.76 vs. 28.58 ± 5.12) (all p < 0.001) compared to the conventional group. CONCLUSIONS: The use of 70-kVp tube voltage combined with DLIR-H for CCTA in normal size patients significantly reduces radiation dose and contrast dose while further improving image quality compared with the conventional 120-kVp tube voltage with 60%ASIR-V. KEY POINTS: • The combination of 70-kVp tube voltage and high-strength deep learning image reconstruction (DLIR-H) algorithm protocol reduces approximately 50% of radiation and contrast doses in coronary computed tomography angiography (CCTA) compared with the conventional scan protocol. • CCTA of normal size (BMI < 26 kg/m2) patients acquired at sub-mSv radiation dose and 24 mL contrast dose through the combination of 70-kVp tube voltage and DLIR-H algorithm achieves excellent diagnostic image quality with a good inter-rater agreement. • DLIR-H algorithm shows a higher capacity of significantly reducing image noise than adaptive statistical iterative reconstruction algorithm in CCTA examination.

Eicosapentaenoic acid mitigates palmitic acid-induced heat shock response, inflammation and repair processes in fish intestine.

Understanding the metabolic effects of fatty acids on fish intestine is critical to the substitution of fish oil with vegetable oils in aquaculture. In this study, the effects of eicosapentaenoic acid (EPA) and palmitic acid (PA) on fish intestine were evaluated in vitro and in vivo. As the first step for in vitro study, an intestinal cell line (SPIF) was established from silver pomfret (Pampus argenteus). Thereafter, the effects of EPA and PA on cell viability, prostaglandin E2 (PGE2) production, and the expression of genes related to heat shock response, inflammation, extracellular matrix (ECM) formation and degradation were examined in SPIF cells. Finally, these metabolic effects of EPA and PA on the intestine were examined in zebrafish (Danio rerio) larvae. Results showed that all tested fatty acids (PA, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, and docosahexaenoic acid) except EPA reduced SPIF viability to distinct degrees at the same concentrations. PA decreased SPIF viability accompanied by an increase in PGE2 level. Meanwhile, PA increased the expression of genes related to heat shock response (grp78, grp94, hsp70, and hsp90) and inflammation (nf-κb, il-1ß, and cox2). Furthermore, PA reduced the expression of collagen type I (col1a1a and col1a1b) and extracellular matrix (ECM) degradation-related gene mmp2, while up-regulating timp2 mRNA expression. In vivo, PA also increased hsp70, il-1ß, and cox2 mRNA levels and limited the expression of collagen type I in the larval zebrafish intestine. Interestingly, the combination of EPA and PA partially recovered the PA-induced changes in cell viability, PGE2 production, and mRNA expression in vitro and in vivo. These results suggest that PA may result in heat shock and inflammatory responses, as well as alter ECM formation and degradation in fish intestine, while EPA could at least partially mitigate these negative effects caused by PA.

First report of Botryosphaeria dothidea causing leaf spot of Camellia oleifera in China.

Camellia oleifera Abel., a small evergreen tree or shrub, is mainly distributed in central and southern China with a larger scale of 4.5 × 106 hectares (Zhu 2020). In May 2021, severe leaf spots were observed in plantation located in Shuangfeng County (27°41'36" N, 111°56'60" E), Hunan Province, China. More than 60 C. oleifera plants were surveyed with over 80% disease incidence. The symptoms on leaves were initially small brown lesions from leaf margins or tips, developing to suborbicular or irregular-shaped dark brown lesions, leading to leaves withered. A total of 60 symptomatic samples were randomly collected. Lesion margins were surface sterilized in 2% sodium hypochlorite for 1 min, rinsed with sterile distilled water for three times, dried, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. Three isolates of Botryosphaeria dothidea were obtained. Colonies of B. dothidea were initially white gradually turning dark-gray with dense aerial mycelium after 6 days. To induce sporulation, colonies of YCB17 were transferred to synthetic nutrient-poor agar (SNA) with sterilized leaves of C. oleifera. Cultures were initially incubated at 25°C in the dark for 3 days, then alternatively exposed to 12-hours near-UV light and 12-hours white light (CHU et al. 2021). After 5 days, conidia formed on leaves were examined microscopically. The conidia were unicellular, aseptate, hyaline, and fusoid, 20.9-25.5×4.7-6.4 µm (n = 50). Morphological characteristics of the isolates matched the description of B. dothidea (Slippers et al. 2014). DNA sequence was amplified using primer pairs ITS1/ITS4 (Tang et al. 2022), EF1-728F/986R (Slippers et al. 2004), and ßt2a/2b (Glass & Donaldson. 1995) respectively. The sequences of three isolates (YCB2, YCB3, YCB17) were deposited in GenBank with accession numbers ON714603, MZ613350, MZ613349 (ITS), OM328342, OM328343, OM328344 (TEF-1α), and OM328345, OM328346, OM328347 (TUB2). A blast search of sequences showed the ITS, TEF-1α, and TUB2 sequences had >99% identity with homologue sequences from B. dothidea isolates IRNHM-KZ49 (MG198191.1), CAP288 (EF638732.1) and Mu1 (MK423987.1), respectively. For pathogenicity testing, healthy leaves of 2-year-old C. oleifera plants in the greenhouse were spray-inoculated with conidial suspension (2×106 conidia/mL) from YCB17. Ten surface-sterilized and wounded leaves per plant were sprayed with 30 µL suspension. The other ten wounded leaves sprayed with sterile distilled water served as control. All plants were kept in the greenhouse with temperature at 26 ± 2°C and 50% relative humidity. After 12 days, initial symptoms were observed on more than 80% leaves inoculated with conidial suspension, whereas no symptoms were observed on the control leaves. The test was repeated three times with similar results. It was found that B. dothidea could cause leaf spot of C. oleifera. The infected leaves showed same symptom as samples. Re-isolated fungi from infected leaves were morphologically identical to B. dothidea. Botryosphaeria dothidea has been reported causing leaf spot in a wide range of hosts, but has not previously been reported causing disease on C. oleifera. To our knowledge, this is the first report of B. dothidea causing leaf spot of Camellia oleifera in China. The information on identification of this fungus may be helpful to the control and prevention of the disease. References: 1. Chu Rui-Tian, et al. 2021. Mycosystema 40(3): 473. 2. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61: 1323. 3. Slippers, B., et al. 2004. Mycologia 96:83. 4. Slippers, B., et al. 2014. Persoonia 33:155. 5. Tang, Y., et al. 2022. Plant Dis. 106: 765. 6. Zhu P.X. People's Daily. 2020.11.09. http://gz.people.com.cn/n2/2020/1119/c194844-34425098.html. *Indicates the corresponding author. Kaifa Guo, E-mail: andygkf@126.com.

Effects of unilateral vs. bilateral resistance training interventions on measures of strength, jump, linear and change of direction speed: a systematic review and meta-analysis.

Exercises can be categorized into either unilateral or bilateral movements. Despite the topic popularity, the answer to the question as to which (unilateral or bilateral) is superior for a certain athletic performance enhancement remains unclear. To compare the effect of unilateral and bilateral resistance training interventions on measures of athletic performance. Keywords related with unilateral, bilateral and performance were used to search in the Web of Science, PubMed databases, and Google Scholar and ResearchGate™ websites. 6365 articles were initially identified, 14 met the inclusion criteria and were included in the final analysis, with overall article quality being deemed moderate. The quantitative analysis comprised 392 subjects (aged: 16 to 26 years). Sub-group analysis showed that unilateral exercise resistance training resulted in a large effect in improving unilateral jump performance compared to bilateral training (ES = 0.89 [0.52, 1.26]). In contrast, bilateral exercise resistance training showed a small effect in improving bilateral strength compared to unilateral (ES = -0.43 [-0.71, -0.14]). Non-significant differences were found in improving unilateral strength (ES = 0.26 [-0.03, 0.55]), bilateral jump performance (ES = -0.04 [-0.31, 0.23]), change of direction (COD) (ES = 0.31 [-0.01, 0.63]) and speed (ES = -0.12 [-0.46, 0.21]) performance. Unilateral resistance training exercises should be chosen for improving unilateral jumping performance, and bilateral resistance training exercises should be chosen for improving bilateral strength performance.