Novel Digital SERS-Microfluidic Chip for Rapid and Accurate Quantification of Microorganisms.

In this work, we present a simple and novel digital surface-enhanced Raman spectroscopy (SERS)-microfluidic chip designed for the rapid and accurate quantitative detection of microorganisms. The chip employs a high-density inverted pyramid microcavity (IPM) array to separate and isolate microbial samples. The presence or absence of target microorganisms is determined by scanning the IPM array using SERS and identifying the characteristic Raman bands. This approach allows for the "digitization" of the SERS response of each IPM, enabling quantification through the application of mathematical statistical techniques. Significantly, precise quantitative detection of yeast was achieved within a concentration range of 106-109 cells/mL, with the maximum relative standard deviation from the concentration calibrated by the cultivation method being 5.6%. This innovative approach efficiently addresses the issue of irregularities in SERS quantitative detection, which arises due to fluctuations in SERS intensity and poor reproducibility. We strongly believe that this digital SERS-microfluidic chip holds immense potential for diverse applications in the rapid detection of various microorganisms, including pathogenic bacteria and viruses.

Carbon sinks/sources' spatiotemporal heterogeneity and their spatial response to economic-social factors in relatively developed regions: A case of China's coastal area.

With the global climate change, carbon reduction in economically active regions has gradually become a focus of attention and its underlying drivers were essential for understanding alterations in ecosystems in response to human behavior. However, the exploration of Carbon Sinks/Sources Patterns (CSSP) in an Economic-Social context was lacking. Distinguished from traditional Net Ecosystem Productivity (NEP) estimation methods, we optimized model parameters, adjusted estimation logic, and revealed CSSP more reasonably. Moreover, spatial econometric model was used to reveal the spatial effects mechanism of Economic-Social Development on CSSP. Over the past 20 years, we revealed that: (a) The pattern of NEP exhibited distinct spatial heterogeneity, with higher sinks observed in the north and offshore regions. It demonstrated regular cyclic fluctuations, averaging a 3-4-year cycle, featuring a gradual ascent followed by a rapid descent; (b) The Carbon Sequestration Capacity (CSC) of vegetation significantly increased. Based on the carbon sink properties, the study area was distinctly divided into three clusters; (c) CSSP have been profoundly affected by economic-social factors. Economic growth and industrial structure optimization contributed to the enhancement of CSC, but population aggregation and urban expansion had negative impacts. The direct effect of innovation capacity and the spatial spillover effect of industrial structure optimization were negative. Overall, exploring CSSP against the backdrop of economic-social factors not only provides a new perspective for understanding the regularities of change and the underlying mechanisms driven by human factors but also offers valuable insights for achieving sustainable development and green growth in other coastal regions globally.

Electrophile-Controlled Regiodivergent Palladium-Catalyzed Imidoylative Spirocyclization of Cyclic Alkenes.

An intermolecular controllable Pd-catalyzed spirocyclization of isocyano cycloalkenes has been developed, offering efficient and selective approaches toward spirocyclic hydropyrrole scaffolds. 2-Azaspiro-1,7-dienes could be obtained through a "chain-walking" process with aryl/vinyl iodides as electrophiles, while the normal Heck product 2-azaspiro-1,6-dienes were selectively generated when aryl triflates were used as the coupling partner of isocyanides. Mechanistic studies suggested that the counteranion of the Pd(II) intermediate played a crucial role in the regioselectivity control. Dihydropyrrole-fused 5,6,7-membered spirocycles were switchably accessed under mild conditions with wide functional group tolerance.

Single-molecule magnet behavior in heterometallic decanuclear [Ln2Fe8] (Ln = Y, Dy, Ho, Tb, Gd) coordination clusters.

Five decanuclear lanthanide-iron clusters, formulated as [Ln2Fe8(hmp)10(µ2-OH)4(µ3-OH)2(µ4-O)4(H2O)6]·6ClO4·xH2O (x ≈ 8, Ln = Y for 1; x ≈ 6, Ln = Dy for 2; x ≈ 6, Ln = Ho for 3; x ≈ 7, Ln = Tb for 4; x ≈ 7, Ln = Gd for 5, Hhmp = 2-(hydroxymethyl)pyridine), have been synthesized and structurally characterized. Single-crystal structural analysis reveals that the cluster consists of six face-sharing defective cubane units. Dynamic magnetic investigations indicated that cluster 2 exhibits single-molecule magnet behavior under a zero dc field eliciting an effective energy barrier of Ueff = 17.76 K and a pre-exponential factor of τ0 = 7.93 × 10-8 s. Investigation of the performance of a series of FeIII-DyIII SMMs indicates that the relatively low energy barrier in 2 is associated with the weak ferromagnetic coupling between FeIII and DyIII ions, while the strength of ferromagnetic interaction in these clusters is mainly related to the bond distances between DyIII and O atoms coordinated to FeIII ions. Clusters 3 and 4 exhibit similar dual relaxation pathways under their respective optimal external applied dc field, where the direct relaxation process occurs in the low-frequency area, which impedes the extraction of the Ueff, while the secondary relaxation process appears at a higher frequency, which is probably a connection with intermolecularly driven relaxation. Our findings offer a magneto-structural correlation model for further investigating the single-molecule magnet behavior in lanthanide-iron systems.

Three-Component Synthesis of Multiple Functionalized Allenes via Copper/Photoredox Dual Catalyzed 1,4-Alkylcyanation of 1,3-Enynes.

Efficient access to multiple functionalized allenes via a three component 1,4-alkylcyanation of enynes with cyclic alcohol derivatives in the presence of trimethylsilyl cyanide (TMSCN) under copper/photoredox dual catalysis has been developed. Both easily transformable aldehyde and cyano groups were introduced to tetra-substituted allenes through light-induced C-C bond cleavage of cyclic butanol and pentanol derivatives. The reactions proceeded smoothly under mild conditions with broad functional groups tolerance.

Palladium-catalysed aryl/monofluoroalkylation of allenamides: access to fluoroalkyl indoles and isoquinolones.

A palladium catalysed construction of fluoroalkyl indoles and isoquinolones through aryl/monofluoroalkylation of allenamides has been developed. Monofluoromethyl-substituted heterocycles could be accessed under mild conditions with broad functional group tolerance. In addition, indole-oxindole bisheterocyclic scaffolds bearing a fluorine atom were successfully synthesized with 3-fluoro-oxindole as the nucleophile by applying this method.

Accumulation of the bitter substance quercetin mediated by the overexpression of a novel seed-specific gene FtRDE2 in Tartary buckwheat.

Tartary buckwheat (Fagopyrum tataricum) is frequently employed as a resource to develop health foods, owing to its abundant flavonoids such as rutin. However, the consumption of Tartary buckwheat (TB) is limited in food products due to the strong bitterness induced by the hydrolysis of rutin into quercetin. This transformation is facilitated by the degrading enzyme (RDE). While multiple RDE isoenzymes exist in TB, the superior coding gene of FtRDEs has not been fully explored, which hinders the breeding of TB varieties with minimal bitterness. Here, we found that FtRDE2 is the most abundant enzyme in RDE crude extracts, and its corresponding gene is specifically expressed in TB seeds. Results showed that FtRDE2 has strong rutin hydrolysis activity. Overexpression of FtRDE2 not only significantly promoted rutin hydrolysis and quercetin accumulation but also dramatically upregulated genes involved in the early phase of flavonoid synthesis (FtPAL1、FtC4H1、Ft4CL1, FtCHI1) and anthocyanin metabolism (FtDFR1). These findings elucidate the role of FtRDE2, emphasizing it as an endogenous factor contributing to the bitterness in TB and its involvement in the metabolic regulatory network. Moreover, correlation analysis revealed a positive relationship between the catalytic activity of RDE extracts and the expression level of FtRDE2 during seed germination. In summary, our results suggest that FtRDE2 can serve as a promising candidate for the molecular breeding of a TB variety with minimal bitterness.

Tartary buckwheat rutin: Accumulation, metabolic pathways, regulation mechanisms, and biofortification strategies.

Rutin is a significant flavonoid with strong antioxidant property and various therapeutic effects. It plays a crucial role in disease prevention and human health maintenance, especially in anti-inflammatory, antidiabetic, hepatoprotective and cardiovascular effects. While many plants can synthesize and accumulate rutin, tartary buckwheat is the only food crop possessing high levels of rutin. At present, the rutin content (RC) is regarded as the key index for evaluating the nutritional quality of tartary buckwheat. Consequently, rutin has become the focus for tartary buckwheat breeders and has made considerable progress. Here, we summarize research on the rutin in tartary buckwheat in the past two decades, including its accumulation, biosynthesis and breakdown pathways, and regulatory mechanisms. Furthermore, we propose several strategies to increase the RC in tartary buckwheat seeds based on current knowledge. This review aims to provide valuable references for elevating the quality of tartary buckwheat in the future.

Icariin alleviates the injury of Sertoli cell junction function by upregulating PKR pathway via ERα/c-fos signaling in aged mice.

ETHNOPHARMACOLOGICAL RELEVENACE: Sertoli cells are vital to maintain spermatogenesis and their function decline during aging. Epimedium has the effects of tonifying kidney-yang, strengthening bones and muscles, and expelling wind and dampness, and is commonly used in the treatment of kidney-yang deficiency, impotence and spermatorrhea. Icariin is the main active ingredients from Epimedium exhibiting delaying aging effects and improving male reproductive dysfunction. Whereas, it remains poorly understood how icariin alleviates age-associated decline in testicular function by protecting against the damage of junction function of Sertoli cells. AIM OF THE STUDY: This study aimed to evaluate the improvement effect of icariin on Sertoli cell junction function damage and explore the underlying mechanisms. MATERIALS AND METHODS: Male C57BL/6 mice and mouse Sertoli cell line TM4 cells were utilized to assess the improvement effect of icariin on aging-associated Sertoli cell junction function injury. H&E staining, transmission electron microscopy, qPCR, Western blot, molecular docking, siRNA transfection, and immunofluorescence were performed in this study. RESULTS: Dietary administration of icariin remarkly attenuated age-associated deterioration in spermatogenic function as evidenced by elevated testicular weight and index, sperm concentration and sperm viability. In addition, icariin protected Sertoli cell junction function from age-associated damage as proven by increased Sertoli cell numbers, improved tight junction ultrastructure, and upregulated junction-related proteins (ZO-1, Occludin and ß-Catenin). Moreover, icariin significantly upregulated ERα/c-fos signaling and PKR pathway in testicular Sertoli cells. Similarly, in vitro studies revealed that deletion of ERα, c-fos or PKR abolished the improvement effects of icariin on Sertoli cell junction function damage. CONCLUSIONS: Icariin effectively mitigates age-associated decline in testicular function by diminished Sertoli cell junction function damage through upregulating PKR pathway via ERα/c-fos signaling. Therefore, attenuating Sertoli cell junction function injury by the upregulation of PKR pathway via ERα/c-fos signaling probably indicates an effective target for the prevention and treatment of testicular spermatogenic function with aging.

Regulatory Module FtMYB5/6-FtGBF1-FtUFGT163 Promotes Rutin Biosynthesis in Tartary Buckwheat.

Tartary buckwheat is highly valued for its abundant rutin (quercetin 3-O-rutinoside). As a flavonoid glycoside, rutin is synthesized with the crucial involvement of UDP-dependent glycosyltransferases (UGTs). However, the functions and transcriptional regulation of the UGT-encoded genes remain poorly understood. This study identified a key gene, FtUFGT163, potentially encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase in Tartary buckwheat through omics analysis and molecular docking methods. The recombinant FtUFGT163 expressed in Escherichia coli demonstrated the capacity to glycosylate isoquercetin into rutin. Overexpression of FtUFGT163 significantly enhanced the rutin content in Tartary buckwheat. Further investigation identified a novel bZIP transcription factor, FtGBF1, that enhances FtUFGT163 expression by binding to the G-box element within its promoter, thereby augmenting rutin biosynthesis. Additional molecular biology experiments indicated that the specific positive regulator of rutin, FtMYB5/6, could directly activate the FtGBF1 promoter. Collectively, this study elucidates a novel regulatory module, termed "FtMYB5/6-FtGBF1-FtUFGT163", which effectively coordinates the biosynthesis of rutin in Tartary buckwheat, offering insights into the genetic enhancement of nutraceutical components in crops.

Interdigital-type antifungal socks for prevention and treatment of tinea pedis.

BACKGROUND: Interdigital tinea pedis is the most common type of foot infection, which is often treated by topical or systemic antifungals. Due to the increase in antifungal resistance, antifungal socks are becoming potential alternatives for the daily management of tinea pedis. METHODS: In this study, antifungal fibres were adopted to produce interdigital hygiene socks to split the third and fourth toe seams of the feet. In vitro antifungal activity was first examined to verify the effectiveness of the socks. Preventive efficacy against tinea pedis was then evaluated among healthy participants, followed by therapeutic effect detection in patients diagnosed with tinea pedis by analysing the improvement in total symptom scores (TTS). RESULTS: The interdigital-type hygiene socks exhibited apparent antifungal activities in vitro. An in vivo study demonstrated significant preventive effects against tinea pedis for interdigital socks compared to plain socks (P = 0.011) and a lower TTS than noninterdigital (P = 0.04) or plain socks (P < 0.0001). Moreover, interdigital socks showed a total effectiveness rate of 72.9% in patients with tinea pedis, with most of the symptoms alleviated. CONCLUSION: Interdigital-type hygiene socks not only exhibited in vitro antifungal activities but also showed significant prophylactic and therapeutic effects against interdigital tinea pedis in vivo.

Enhancing rutin accumulation in Tartary buckwheat through a novel flavonoid transporter protein FtABCC2.

Tartary buckwheat (Fagopyrum tataricum) is an annual coarse cereal from the Polygonaceae family, known for its high content of flavonoid compounds, particularly rutin. But so far, the mechanisms of the flavonoid transport and storage in Tartary buckwheat (TB) remain largely unexplored. This study focuses on ATP-binding cassette transporters subfamily C (ABCC) members, which are crucial for the biosynthesis and transport of flavonoids in plants. The evolutionary and expression pattern analyses of the ABCC genes in TB identified an ABCC protein gene, FtABCC2, that is highly correlated with rutin synthesis. Subcellular localization analysis revealed that FtABCC2 protein is specifically localized to the vacuole membrane. Heterologous expression of FtABCC2 in Saccharomyces cerevisiae confirmed that its transport ability of flavonoid glycosides such as rutin and isoquercetin, but not the aglycones such as quercetin and dihydroquercetin. Overexpression of FtABCC2 in TB hairy root lines resulted in a significant increase in total flavonoid and rutin content (P < 0.01). Analysis of the FtABCC2 promoter revealed potential cis-acting elements responsive to hormones, cold stress, mechanical injury and light stress. Overall, this study demonstrates that FtABCC2 can efficiently facilitate the transport of rutin into vacuoles, thereby enhancing flavonoids accumulation. These findings suggest that FtABCC2 is a promising candidate for molecular-assisted breeding aimed at developing high-flavonoid TB varieties.

Innovative approach for high-throughput exploiting sex-specific markers in Japanese parrotfish Oplegnathus fasciatus.

BACKGROUND: The use of sex-specific molecular markers has become a prominent method in enhancing fish production and economic value, as well as providing a foundation for understanding the complex molecular mechanisms involved in fish sex determination. Over the past decades, research on male and female sex identification has predominantly employed molecular biology methodologies such as restriction fragment length polymorphism, random amplification of polymorphic DNA, simple sequence repeat, and amplified fragment length polymorphism. The emergence of high-throughput sequencing technologies, particularly Illumina, has led to the utilization of single nucleotide polymorphism and insertion/deletion variants as significant molecular markers for investigating sex identification in fish. The advancement of sex-controlled breeding encounters numerous challenges, including the inefficiency of current methods, intricate experimental protocols, high costs of development, elevated rates of false positives, marker instability, and cumbersome field-testing procedures. Nevertheless, the emergence and swift progress of PacBio high-throughput sequencing technology, characterized by its long-read output capabilities, offers novel opportunities to overcome these obstacles. FINDINGS: Utilizing male/female assembled genome information in conjunction with short-read sequencing data survey and long-read PacBio sequencing data, a catalog of large-segment (>100 bp) insertion/deletion genetic variants was generated through a genome-wide variant site-scanning approach with bidirectional comparisons. The sequence tagging sites were ranked based on the long-read depth of the insertion/deletion site, with markers exhibiting lower long-read depth being considered more effective for large-segment deletion variants. Subsequently, a catalog of bulk primers and simulated PCR for the male/female variant loci was developed, incorporating primer design for the target region and electronic PCR (e-PCR) technology. The Japanese parrotfish (Oplegnathus fasciatus), belonging to the Oplegnathidae family within the Centrarchiformes order, holds significant economic value as a rocky reef fish indigenous to East Asia. The criteria for rapid identification of male and female differences in Japanese parrotfish were established through agarose gel electrophoresis, which revealed 2 amplified bands for males and 1 amplified band for females. A high-throughput identification catalog of sex-specific markers was then constructed using this method, resulting in the identification of 3,639 (2,786 INS/853 DEL, ♀ as reference) and 3,672 (2,876 INS/833 DEL, ♂ as reference) markers in conjunction with 1,021 and 894 high-quality genetic sex identification markers, respectively. Sixteen differential loci were randomly chosen from the catalog for validation, with 11 of them meeting the criteria for male/female distinctions. The implementation of cost-effective and efficient technological processes would facilitate the rapid advancement of genetic breeding through expediting the high-throughput development of sex genetic markers for various species. CONCLUSIONS: Our study utilized assembled genome information from male and female individuals obtained from PacBio, in addition to data from short-read sequencing data survey and long-read PacBio sequencing data. We extensively employed genome-wide variant site scanning and identification, high-throughput primer design of target regions, and e-PCR batch amplification, along with statistical analysis and ranking of the long-read depth of the variant sites. Through this integrated approach, we successfully compiled a catalog of large insertion/deletion sites (>100 bp) in both male and female Japanese parrotfish.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

Corneal differences between healthy and subclinical patients assessed using two diferente corneal tomographers / Diferenças entre as córneas de pacientes saudáveis e pacientes subclínicos avaliados com dois tipos de tomógrafos de córnea

ABSTRACT Purpose: To analyze subclinical keratoconus topography indexes using Pentacam and Orbscan-II measurements to identify evidences for seeking sensitive indexes to screen and diagnose subclinical keratoconus. Methods: Fifty healthy participants (50 eyes) and 40 patients with subclinical keratoconus (40 eyes) were included. Seven common parameters including corneal thickness at the thinnest point; minimum curvature of the front surface (minimum simulated keratometry value, SimK's Min); maximum curvature of the front surface (maximum simulated keratometry value, SimK's Max); the frontal corneal surface best-fit spherical radius of the curvature; the back corneal surface best-fit spherical radius of curvature; the anterior corneal surface height (anterior Diff value); and the posterior corneal surface height (posterior Diff value) measured by Pentacam and Orbscan-II between normal and subclinical keratoconus eyes were compared. Results: Statistical differences between the healthy and subclinical keratoconus groups (p<0.01) were found in all corneal parameters measured using both devices. Differences in the minimum curvature of the front surface (SimK's Min), thinnest point, anterior Diff value, and posterior Diff value were significant between Pentacam and Orbscan-II in the subclinical keratoconus group (p<0.05). Conclusion: The findings of this study identify the differences between normal and subclinical keratoconus eyes at the minimum curvature of the front surface, maximum curvature of the front surface, frontal corneal surface best-fit spherical radius of curvature, back corneal surface best-fit spherical radius of curvature, Anterior Diff value, and Posterior Diff value measures using Orbscan II and Pentacam that can help eye care practitioners clinically diagnose subclinical keratoconus.

RESUMO Objetivo: Analisar os índices subclínicos de to pografia de ceratocone utilizando as medidas feitas com Pentacam e com Orbscan-II para identificar evidências para a busca de índices sensíveis para triagem e diagnóstico de ceratocone subclínico. Métodos: Cinquenta participantes saudáveis (50 olhos) e 40 pacientes com ceratocone subclínico (40 olhos) foram incluídos. Sete parâmetros comuns, incluindo a espessura da córnea no ponto mais fino; a curvatura mínima da superfície frontal (valor mínimo da ceratometria simulada, Min de SimK); a curvatura máxima da superfície frontal (valor máximo da ceratometria simulada, Max de SimK); a superfície frontal e a superfície posterior da córnea de melhor ajuste ao raio da curvatura, a altura da superfície anterior da córnea (valor Diff anterior) e a altura da superfície corneana posterior (valor Diff posterior) medidos pelo Pentacam e pelo Orbscan-II entre os olhos normais e com ceratocone subclínico foram comparados. Resultados: As diferenças estatísticas entre os grupos saudável e com ceratocone subclínico (p<0,01) foram encontradas em todos os parâmetros corneanos medidos usando ambos os dispositivos. Diferenças na curvatura mínima da superfície frontal (Min de SimK) no ponto mais fino, no valor Diff anterior e no valor Diff posterior foram significativas entre Pentacam e Orbscan-II no grupo com ceratocone subclínico (p<0,05). Conclusão: Os achados deste estudo identificam as diferenças entre olhos normais e com ceratocone subclínico para a curvatura mínima da superfície frontal, a curvatura máxima da superfície frontal, a superfície corneana frontal e a superfície corneana posterior de melhor ajuste ao raio esférico da curvatura e as medidas de Diff anterior e posterior usando Orbscan II e o Pentacam que podem auxiliar os profissionais de oftalmologia a diagnosticar clinicamente o ceratocone subclínico.