The purity of commercial cucumber varieties, predominantly F1 hybrids, is a critical factor in seed quality assessment. While SSR or SNP markers are commonly used, insertion-deletion (InDel) markers offer greater stability and practical advantages, including lower cost, compatibility with agarose gel electrophoresis, and suitability for multiplex PCR. In this study, we developed long InDel (> 30bp) markers for variety purity detection in cucumber using whole-genome resequencing data from 182 accessions. A total of 326,479 InDel loci were identified, of which 2655 long InDels satisfy the following requirements which have conserved 200bp flanking sequences, high polymorphism and suitability for primer design. From these, 74 markers with minor allele frequency (MAF) > 0.2 across four cucumber populations were evenly distributed across seven chromosomes and synthesized. All markers successfully amplified target products, and 10 exhibited high heterozygosity and clear bands on agarose gels. These 10 markers were further adapted for multiplex capillary electrophoresis detection by adding different fluorescent groups. DNA fingerprints of 66 commercial cucumber varieties were constructed using these markers. Among them, four markers InD_Cu35, InD_Cu46, InD_Cu61 and InD_Cu66, showed at least one heterozygous genotype across all varieties, making them particularly suitable for purity testing of cucumber hybrid varieties. The practical utility of these markers was confirmed by assessing the purity of a new hybrid variety, Lvlinglong (98.9%). These long InDel markers provide a robust, flexible, and cost-effective tool for routine cucumber variety purity detection and genetic analysis.

Forensic microbiology allows identification by microbiome analysis when human DNA analysis is limited. However, microbiome-based approaches have not been widely used in forensic medicine. The spatial and temporal stability of microbial communities in response to environmental exposures represents a serious challenge. This study investigates the dynamics of skin and saliva microbial markers in response to environmental exposure to advance their forensic applicability. We selected 8 characteristic microorganisms (Finegoldia magna, Corynebacterium tuberculostearicum, Cutibacterium acnes for skin; Haemophilus parainfluenzae, Streptococcus oralis, Prevotella melaninogenica for saliva; Achromobacter, Pseudomonas as environmental markers) and developed two multiplex amplification systems for capillary electrophoresis detection. A longitudinal exposure model (0-120 days) under controlled indoor and dry environment was applied to 420 skin and saliva samples from 10 subjects. Saliva samples exhibited significant microbial community shifts between environments (R2 = 0.4137). Dry environment preserved community structure better, evidenced by delayed inflection points in markers' proportions and stronger clustering in PCoA. Redundancy analysis correlated microbial markers with temperature and humidity, and this correlation varied. The microbial markers remained detectable after 120 days of environmental exposure and were able to provide information on community characteristics and deposition time. This study explored the effects of environment and time on microbial communities and the response patterns of microbial markers to temperature and humidity. It is expected to provide help for forensic microbial research related to environmental exposure.

ObjectiveThis study aimed to investigate the prevalence and etiological profile of decreased red blood cell osmotic fragility among voluntary blood donors in Shenzhen, China, and to assess the public health implications for donor screening policies and transfusion safety in thalassemia-endemic regions.MethodsThis cross-sectional study enrolled 362 voluntary blood donors at the Shenzhen Blood Center from July to September 2025. All donors underwent initial hemoglobin screening and red blood cell osmotic fragility testing. Donors with decreased fragility received comprehensive evaluation including complete blood count, serum ferritin measurement, hemoglobin electrophoresis, and thalassemia gene detection. Transfusion efficacy was preliminarily assessed by analyzing recipient hemoglobin increments.ResultsAmong the 14 donors (3.87%) with decreased red blood cell osmotic fragility, 9 (64.3%) were confirmed as thalassemia gene carriers (7 with α-thalassemia, 1 with β-thalassemia, and 1 with compound α/β-thalassemia), and 7 (50.0%) had biochemical iron deficiency (ferritin <30 ng/mL), with 4 individuals meeting both criteria, while 2 donors had neither thalassemia nor iron deficiency. Blood components from donors with decreased osmotic fragility, including those from thalassemia carriers, demonstrated effective hemoglobin recovery in recipients, with post-transfusion increments observed in all transfused cases.ConclusionIn Southern China, decreased red blood cell osmotic fragility among blood donors is primarily associated with thalassemia trait and iron deficiency. These findings highlight the need for targeted screening strategies and evidence-based policies to optimize donor health management while ensuring blood safety and availability. The study provides a public health framework for managing subclinical hematological conditions in blood donor populations in endemic regions.

DNA mismatch repair (MMR)/microsatellite instability (MSI) status is an indispensable biomarker for predicting immunotherapy response, screening for Lynch syndrome (LS), and molecularly classifying endometrial carcinoma (EC). To investigate the consistency and the causes of discordance among different methods for MMR/MSI, we performed MMR immunohistochemistry (MMR-IHC) and next-generation sequencing (NGS) for MSI status (MSI-NGS). Formalin-fixed and paraffin-embedded (FFPE) specimens were collected from 141 EC patients. 38 (26.95%) patients exhibited deficient MMR (dMMR), while 103 (73.05%) harboured proficient MMR (pMMR). There were no significant differences in ages or histological types between the dMMR and pMMR groups (all P > 0.05). Molecular classification based on NGS results revealed no significant differences in ages among the POLE-mutated (POLE mut), MSI-high (MSI-H), p53-abnormal (p53 abn), and no specific molecular profile (NSMP) subtypes (all P > 0.05). The proportion of aggressive histological subtypes in the MSI-H group (44.0%, 11/25) was higher than that in the NSMP group (17.4%, 15/86; P < 0.01); lower than that in the p53 abn group (80.0%, 12/15; P < 0.05); but there was no difference compared to the POLE mut group (33.3%, 5/15; P > 0.05). MSI-NGS classified 114 (80.85%) cases as microsatellite stable (MSS) phenotype and 27 (19.15%) as MSI-H phenotype. Compared to MMR-IHC, discordance was observed in 11 cases between the two methodologies, and the consistency rate was 92.2% (130/141). Among the inconsistent 11 cases, PCR-capillary electrophoresis (PCR-CE) detection showed 2 MSI-H cases, 3 MSI-low (MSI-L), and 6 MSS. Among them, 6 cases with concurrent loss of MLH1 and PMS2 exhibited MLH1 promoter hypermethylation and lacked MMR gene mutations. Among the 38 dMMR patients, 23.7% (9/38) were diagnosed as LS with the germline mutations in MMR genes, and all of their MSI-NGS results were MSI-H. Two dedifferentiated EC (DEC) cases were detected with MLH1 promoter hypermethylation, accompanied by concurrent loss of MLH1 and PMS2, with tumor mutational burden-high (TMB-H) and no MMR gene mutations. Among the 7 (4.96%) patients with isolated MSH6 deficiency, MSI-NGS analysis revealed 3 MSS cases and 4 MSI-H, while PCR-CE detection revealed 5 MSI-H cases. Among the 3 (2.13%) patients with isolated PMS2 deficiency, MSI-NGS identified 2 MSS cases and 1 MSI-H, which were consistent with the PCR-CE results. In conclusion, MMR-IHC reveals relatively high concordance with MSI-NGS. Discordance primarily occurs in the cases with MLH1 promoter hypermethylation and isolated PMS2 or MSH6 deficiency. When necessary, comprehensive analyses should be conducted combining MLH1 gene methylation, PCR-CE testing, MMR gene mutations, and TMB status, so as to provide precise molecular classification for EC patients.

Advances in the detection of azodicarbonamide and the metabolic product semicarbazide.

Analysis of lysozyme in human saliva by CE-UV: A new simple, fast, and reliable method.

Homochiral zeolite imidazolateframework-8 (ZIF-8) combines the inherent characteristics of ZIF-8 with the chiral recognition function and open chiral channel, becoming one preferred choice for enantiomer separation. In this work, ZIF-8 with D-histidine as the chiral carbon center (D-His-ZIF-8) was introduced into the pores to enhance the extraction performance of hollow-fiber liquid-phase microextraction (HF-LPME). A novel method was developed for the selective and sensitive determination of chiral amino acids related to the nervous system by D-His-ZIF-8@HF-LPME coupled with capillary electrophoresis and laser-induced fluorescence detection. Under the optimal conditions, D-type amino acids could obtain higher enrichment efficiency than L-type ones due to the stronger geometry-dependent hydrogen-bonding interactions between L-type amino acids and the chiral channels of ZIF-8 materials modified with D-His, and the maximum enrichment factor reached 640 times (for D-serine). This developed method has been successfully applied to the analyses of four chiral amino acids (D/L-serine and D/L-aspartic acid) in exhaled breath condensate and saliva samples of healthy volunteers. The limits of detection were 0.030-0.30nM (S/N = 3) in the real sample matrixes. This developed method meets the principles of green analytical chemistry and achieves a greenness score of 80 assessed by the analytical eco-scale tool. This proposed method provides one green alternative for the sensitive and accurate analyses of chiral amino acids in the non-invasive body fluids.

Fumarate, succinate, maleate, dihydroxyfumarate, D–tartarate, L–tartarate, DL–tartarate, L-malate, D-malate, oxaloacetate, citrate, and DL-isocitrate in the 5–100 μM concentration range were incubated in 12.5 mM HEPES/25 mM TRIS base containing 200 μM Eu3+–tetracycline and 60% (v/v) formamide (pH unadjusted). After 30 min of incubation, they were separated at 4 °C by capillary electrophoresis utilizing laser-induced luminescence detection with 12.5 mM HEPES/25 mM TRIS base containing 60% formamide as the running buffer. All analytes yielded peaks, with the exception of fumarate, succinate, and maleate. L-Malate was detected down to 100 nM. The main component of this study was the analysis of malate. The objective was to develop a stereoselective methodology for the detection of L-malate. This was achieved by varying the formamide concentration and separation temperature. When the temperature was increased to 22 °C and the formamide concentration decreased to 40%, the sensitivity for L-malate was diminished about 10-fold, but that for D-malate was eliminated. This combination of conditions allowed for the stereospecific analysis of L-malate.

Results of the promise study from ~30,000 individuals screened for monoclonal gammopathies

Digestion supplemented with commercial proteases: Evaluation of the fate of gluten immunogenic peptides in pizza.

High sensitivity and specificity analysis of multiple miRNAs.

蛋白质的特异性检测在生物分析和临床诊断中具有重要意义。现有方法如免疫固定电泳和蛋白质印迹法，需要手工染色和脱色等步骤，操作繁琐，耗时久且定量能力有限。本文结合在线荧光成像技术开发了一种蛋白质免疫聚丙烯酰胺凝胶电泳（PAGE）定量检测方法。该方法首先采用荧光标记抗体结合目标蛋白质，使用甲醛交联免疫复合物后进行电泳和在线成像，最终通过计算凝胶电泳图像中游离抗体的荧光信号强度实现定量分析。整个检测流程可在1.5 h内完成。本文以人转铁蛋白（transferrin，TRF）为模式蛋白进行方法学验证，结果显示该方法的线性范围为5.0~200.0 mg/L，线性相关系数（R2）为0.993 0，制作标准曲线的标准溶液中荧光信号强度的最大相对标准偏差（RSD）为1.65%，检出限为0.5 mg/L，加标回收率为98.2%~105.0%，表明该检测方法具有良好的精密度、灵敏度和准确度。相较于传统的PAGE方法，该方法利用实时荧光成像技术实现了对目标蛋白质的在线定量检测，具有分辨率高、特异性强、操作简便、分析快速以及重现性好等优点。该方法具有普适性，可应用于其他蛋白质的定量检测，在药物制备和临床诊疗等领域具有重要的应用价值。

Multi-fingerprint analysis for interpretation of the quality differences in polysaccharides during Dendrobium huoshanense traditional processing.

Intron 22 inversion (Inv22) of the factor VIII gene (F8) accounts for approximately 45% of severe hemophilia A (HA) cases. Detecting Inv22 has become the primary screening method for severe HA. Currently, agarose gel electrophoresis (AGE) following long-distance polymerase chain reaction (LD-PCR) is commonly used in clinical settings to separate the amplified fragments of Inv22. However, AGE is hindered by lengthy processing times, instability, and inaccuracies in quantifying DNA content and assessing fragment sizes. We combined LD-PCR with capillary gel electrophoresis (CGE) for the identification of Inv22 in HA. Three primers were designed for LD-PCR to differentiate between Inv22, carriers, and wild types. We optimized the reaction system and conditions for CGE to effectively separate the amplified fragments. The optimal dilution ratio and buffer conditions for detecting Inv22 using CGE were 300 × and 0.1 × TE buffer. The ideal voltage and duration were 5.0kV for 80min. Under these conditions, the amplified fragments could be effectively separated, allowing for the direct measurement of concentration and size of the target fragments using ProSize data analysis software. The LD-PCR combined with the CGE assay for detecting Inv22 in F8 within HA populations has been successfully established. This method reduces both the time and labor required for detecting Inv22 in clinical practice, thereby advancing genetic diagnostic technology for hemophilia.

A microchip electrophoresis method for rapid nucleic acid extraction and bacteria detection.

Maize yellow mosaic virus (MaYMV) is an emerging polerovirus that was first reported in maize in China in 2013 but has since been reported in Africa and the Americas. Due to its novelty, no antibodies or serological diagnostics for the virus existed prior to the current study. In this study, we developed the first purification method for maize yellow mosaic virus with the goal of generating an antibody and subsequently developing diagnostic tests. Yields of 0.634-1.275 milligrams of virus per kilogram of maize tissue were obtained, which is comparable to the yields obtained for other grass-infecting poleroviruses. Under an electron microscope, purified virus particles appear icosahedral in shape and roughly 30 nm in diameter. A polyclonal antibody was generated against the gradient-purified virus preparation and cross-adsorbed to uninfected maize tissue. The antibody is effective for detection of MaYMV via enzyme-linked immunosorbent assay (ELISA) and western blot. The ELISA could detect virus in quantities as low as 0.395 µg and in 1:32 dilutions of infected plant extract, and the antibody showed little to no cross-reactivity with five closely related viruses. After protein gel electrophoresis and antibody detection, protein bands corresponding in size to two viral structural proteins could be seen: the coat protein of roughly 21 kDa and the readthrough protein, around 72 kDa. Finally, virus particles purified using this protocol were found to be transmissible by the primary aphid vector, Rhopalosiphum maidis, and to cause systemic, symptomatic infection of maize plants, indicating that the vector transmissibility and infectivity of the virus particles were preserved during purification.

Biarsenical-based fluorescent labeling of metallothioneins as a method for ultrasensitive quantification of poly-Cys targets.

Blanding's turtles (Emydoidea blandingii) are a species of conservation concern throughout their natural range. Headstarting is a common chelonian conservation technique in which neonates are reared in managed-care settings before release, but health assessments are rarely incorporated. From 2020 to 2021 we assessed headstarted turtle health pre-release and 1 mo, 1 yr, and 2 yr after release using physical examination, hematology, plasma biochemistry, protein electrophoresis, and pathogen detection in three Illinois counties. Results were compared to wild-reared juveniles in the same habitats. Overall, 767 assessments from 561 turtles were included. Wild-reared and 2 yr post-release headstarts had higher incidence of hemoparasites, asymmetrical nares, and increased creatine kinase and aspartate aminotransferase activities (P<0.05) compared to all other groups. Erythrocyte sedimentation rate and heterophil:lymphocyte ratio were greater, while total leukocyte and lymphocyte counts were lower (P<0.05) in pre-release headstarts compared to wild-reared juveniles. Total solids, albumin, and beta globulins were higher, while the calcium:phosphorous ratio was lower (P<0.05) in pre-release headstarts and wild-reared juveniles vs. other groups. Bile acid levels were highest in pre-release headstarts (P<0.05). Body condition and gamma globulins increased following release, while alpha globulins and the albumin:globulin ratio decreased following release (P<0.05). Two pre-release and one post-release headstart tested positive for Emydomyces testavorans, one post-release headstart was positive for Mycoplasmopsis sp., and nine post-release turtles were positive for adenoviruses. Overall, rearing conditions have a profound and temporally dynamic impact on Blanding's health assessment parameters. Future studies should evaluate long-term impacts on morbidity and mortality to support positive health status and conservation outcomes.

The article discusses modern approaches to the visualization of nucleic acids after gel electrophoresis, with an emphasis on dyes that are safe for human health and the environment. Traditionally, intercalating fluorescent dyes, particularly ethidium bromide (EtBr), have been used for DNA detection. Despite its high efficiency, EtBr is characterized by significant toxicity, mutagenic activity, and environmental hazards. The article provides a detailed analysis of the harmful effects of EtBr on cells and offers recommendations for the safe handling of this compound, including disposal and personal protective measures. The study also analyzes modern alternative dyes available on the market, such as SYBR Green I/II, GelRed™, GelGreen™, Midori Green, and others, which often demonstrate higher sensitivity and can be used without intense ultraviolet irradiation. Moreover, such compounds do not damage nucleic acids or inhibit enzymatic reactions, which is important for the success of further experimental procedures such as PCR, cloning, or sequencing. A comparative characterization of commercial dyes is provided, including an assessment of their advantages, disadvantages, spectral properties, and cost. Despite a higher purchase price, disposal costs and personnel risks are lower when using modern dyes compared to EtBr. The transition to safer alternatives enhances the efficiency, sensitivity, and safety of laboratory practice in molecular biology and aligns with modern environmental standards. Keywords: detection of nucleic acids, ethidium bromide, fluorescent dyes, gel electrophoresis, labor protection and laboratory safety

This paper introduces a novel contactless single-chip detector that utilizes impedance-to-digital conversion technology to measure impedance in the microfluidic channel or capillary format analytical device. The detector is designed to operate similarly to capacitively coupled contactless conductivity detectors for capillary electrophoresis or chromatography but with the added capability of performing frequency sweeps up to 200 kHz. At each recorded data point, impedance and phase-shift data can be extracted, which can be used to generate impedance versus frequency plots, or phase-shift versus frequency plots. Real and imaginary parts can also be calculated from the data, allowing for the generation of Nyquist diagrams. This detector represents the first of its kind in the contactless conductivity class to provide spectrum-type data, as demonstrated in capillary electrophoresis experiments.