Microbial extracellular vesicles contribute to antimicrobial resistance.

With the escalating global antimicrobial resistance crisis, there is an urgent need for innovative strategies against drug-resistant microbes. Accumulating evidence indicates microbial extracellular vesicles (EVs) contribute to antimicrobial resistance. Therefore, comprehensively elucidating the roles and mechanisms of microbial EVs in conferring resistance could provide new perspectives and avenues for novel antimicrobial approaches. In this review, we systematically examine current research on antimicrobial resistance involving bacterial, fungal, and parasitic EVs, delineating the mechanisms whereby microbial EVs promote resistance. Finally, we discuss the application of bacterial EVs in antimicrobial therapy.

Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

A preliminary exploration for co-detecting RNA virus and STR type on capillary electrophoresis in forensic practice.

RNA virus infection such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection shows severe respiratory symptoms on human and could be an obvious individual characteristic for investigations in forensic science. As for biological samples suspected to contain RNA virus in forensic casework, it requires respective detection of viral RNA and human DNA: reverse transcriptase polymerase chain reaction and DNA type (short tandem repeat [STR] analysis). Capillary electrophoresis (CE) has been shown to be a versatile technique and used for a variety of applications, so we preliminarily explored the co-detection of RNA virus and STR type on CE by developing a system of co-detecting SARS-CoV-2 and STR type under ensuring both the efficiency of forensic DNA analysis and safety of the laboratory. This study investigated the development and validation of the system, including N and ORF1ab primer designs, polymerase chain reaction amplification, allelic ladder, CE detection, thermal cycling parameters, concordance, sensitivity, species specificity, precision, and contrived and real SARS-CoV-2 sample studies. Final results showed the system could simultaneously detect SARS-CoV-2 and STR type, further indicating that CE has possibilities in the multi-detection of RNA viruses/STR type to help to prompt individual characteristics (viral infection) and narrow the scope of investigation in forensic science.

The emerging role of extracellular vesicles in fungi: a double-edged sword.

Fungi are eukaryotic microorganisms found in nature, which can invade the human body and cause tissue damage, inflammatory reactions, organ dysfunctions, and diseases. These diseases can severely damage the patient's body systems and functions, leading to a range of clinical symptoms that can be life-threatening. As the incidence of invasive fungal infections has progressively increased in the recent years, a wealth of evidence has confirmed the "double-edged sword" role of fungal extracellular vesicles (EVs) in intercellular communication and pathogen-host interactions. Fungal EVs act as mediators of cellular communication, affecting fungal-host cell interactions, delivering virulence factors, and promoting infection. Fungal EVs can also have an induced protective effect, affecting fungal growth and stimulating adaptive immune responses. By integrating recent studies, we discuss the role of EVs in fungi, providing strong theoretical support for the early prevention and treatment of invasive fungal infections. Finally, we highlight the feasibility of using fungal EVs as drug carriers and in vaccine development.

Combining the third molar mineralization to further improve the accuracy of the Kvaal's method in dental age estimation of subadults in northern China.

The morphological changes based on deposition of secondary dentin and mineralization of the third molar have been proven to be related to chronological age. However, Kvaal's method on the theory of deposition of secondary dentin was controversial with respect to dental age estimation in the recent research. The aim of this study was to combine the parameters of Kvaal's method with relatively high correlation coefficients and mineralization stages of the third molar to improve the accuracy of predicting the dental age of subadults in northern China. A total of 340 digital orthopantomograms of subadults aged from 15 to 21 years were analysed. A training group was used to test the accuracy of the original Kvaal's method and to establish novel methods for subadults in northern China. A testing group was used to compare the accuracy of the newly established methods with the Kvaal's original method and with published method specifically used in northern China. To increase the feasibility of our estimation model, we combined the mineralization of the third molar to build a combined specific formula. The results showed that the combined specific model increased the coefficient of determination to 0.513, and the standard error of the estimate was reduced to 1.482 years. We concluded that the combined specific model based on the deposition of secondary dentin and mineralization of the third molar could improve the accuracy of dental age assessment of subadults in northern China. Key Points: The decrease in the dental pulp cavity based on deposition of secondary dentin is a useful variable for assessing age.A total of 340 orthopantomographs were used in this research, including 278 in training groups and 62 in testing groups.Original Kvaal's method underestimated the dental age for subadults in northern China.The equation of combined specific method constructed in our study was proved more suitable to calculate dental age for subadults in northern China.

Role of nano-hydrogels coated exosomes in bone tissue repair.

With the development of nanotechnology, nanomaterials are widely applied in different areas. Some nanomaterials are designed to be biocompatible and can be used in the medical field, playing an important role in disease treatment. Exosomes are nanoscale vesicles with a diameter of 30-200 nm. Studies have shown that exosomes have the effect of angiogenesis, tissue (skin, tendon, cartilage, et al.) repair and reconstruction. Nano-hydrogels are hydrogels with a diameter of 200 nm or less and can be used as the carrier to transport the exosomes into the body. Some orthopedic diseases, such as bone defects and bone infections, are difficult to handle. The emergence of nano-hydrogels coated exosomes may provide a new idea to solve these problems, improving the prognosis of patients. This review summarizes the function of nano-hydrogels coated exosomes in bone tissue repair, intending to illustrate the potential use and application of nano-hydrogels coated exosomes in bone disease.

Alignment analysis of Brainlab knee 3 navigation-guided total knee arthroplasty using the adjusted mechanical method.

Purpose: With the application of navigation technology in Total Knee Arthroplasty (TKA), TKA procedures have become various. Studies have shown that navigation can improve the alignment of patients' lower limbs. To verify this conclusion, we collected the clinical data from patients who underwent Brainlab knee 3 navigation-guided TKA. Brainlab knee 3 is a completely new software that takes a different approach to address the current challenges of navigated TKA. During the procedure, we applied the Adjusted Mechanical Alignment (AMA) principle and took soft tissue balance as a priority. We aim to explore the patients' lower limb alignment changes who underwent the Brainlab knee 3 navigation-guided TKA using the AMA method. Methods: Fifty consecutive patients who underwent total knee arthroplasty using the Brainlab knee3 knee navigation system (Smith&Nephew) from January to August 2021 by the same doctor (Yunsu Chen) in the Department of Joint Surgery of the Shanghai Sixth People's Hospital were included. Hip-Knee-Ankle Angle (HKAA), anatomic Femur Tibia Angle (FTA), Lateral Distal Femoral Angle (LDFA), and medial proximal tibia angle (MPTA) were measured on preoperative and postoperative full-length lower-limbs x-ray films or weight-bearing anterior and lateral knee radiographs for observational and descriptive study. The preoperative and postoperative knee alignment changes were analyzed through paired t-test or nonparametric Wilcoxon test using SPSS 25.0 software. Results: Pre-operative and post-operative HKAA both obeyed normal distribution. The mean preoperative HKAA was 169.8° (154.9-178.7°) with a standard deviation of 5.41; the postoperative HKAA was 175.7° (168.4-180.0°) with a standard deviation of 2.81. Using the two-sample paired t-test to analyze, the result showed P = 0.000 < 0.05; a statistically significant difference exists. The preoperative and postoperative FTA obeyed normal distribution as well. The mean preoperative FTA was 174.7° (163.4-179.9°) with a standard deviation of 3.90; postoperative 175.6° (167.0-179.9°) with a standard deviation of 2.77. Using the two-sample paired t-test to analyze, the result showed P = 0.140 > 0.05, the difference was not statistically significant. The preoperative LDFA was normally distributed, while postoperative LDFA was not. The mean preoperative LDFA was 90.7° (83.5-99.6°) with a standard deviation of 3.83; the median of postoperative LDFA was 91.6° (86.0-103.2°) with an interquartile range of 2.93. Using the two-sample paired Wilcoxon test, the result showed P = 0.052 > 0.05; the difference was not statistically significant. Preoperative MPTA obeyed normal distribution, while postoperative MPTA did not. The mean preoperative MPTA was 83.5° (72.7-92.9°), with a standard deviation of 3.66; the median of postoperative MPTA was 89.3° (84.6-95.6°), with an interquartile range of 1.45. Using the two-sample paired Wilcoxon test, the result shows P = 0.000 < 0.05; a statistically significant difference exists. Conclusion: In our study, AMA alignment was applied in Brainlab Knee3 computer navigation-assisted total knee arthroplasty. The femoral and tibial osteotomy angles were minimally adjusted according to soft tissue situations to reduce soft tissue release. We found AMA alignment provides good control of knee alignment in the coronal plane of the lower limbs, which is a reliable technique.

Influence of bacterial community diversity, functionality, and soil factors on polycyclic aromatic hydrocarbons under various vegetation types in mangrove wetlands.

Polycyclic aromatic hydrocarbons (PAHs) are prevalent organic pollutants in coastal ecosystems, particularly in mangrove wetlands. However, it is still largely unclear how PAHs affect the soil bacterial community under various vegetation types in the Greater Bay Area. Here, we selected soil samples from four sites with different vegetation types (native mangrove forest dominated by Kandelia candel, invasive mangrove forest dominated by Sonneratia apetala, unvegetated mudflat, and riverine runoff outlet) in the Qi'ao and Futian Nature Reserves. We investigated the effects of PAHs on soil bacterial community composition and diversity, function, and co-occurrence via 16S rRNA high-throughput sequencing. PAHs obviously reduced soil bacterial community diversity and richness. Based on PICRUSt 2, PAHs demonstrated positive influences on PAHs degradation metabolism related bacterial genes. Meanwhile, we predicted that riverine runoff outlets can potentially degrade PAHs, may donate to sustain healthy mangrove ecosystem. Also, PAHs and total nitrogen (TN) were crucial factors driving the soil bacterial community in Qi'ao sites, whereas in the Futian sites, PAHs and SOC were more important. PAHs, SOC and TN showed negative effects on specific bacteria abundance. Subsequently, environmental factors and PAHs levels influenced the soil bacterial ecological functions community. Co-occurrence network analysis revealed non-random assembly patterns of the bacterial communities. SBR1031 and A4b were the keystone genera and played a crucial role whgich played an irreplaceable role in PAHs degradation in Qi'ao and Futian sites. PAHs inhibited specific microbial activity and metabolism in native mangrove forest, while affects positively to bacterial community in riverine runoff outlet which might profoundly affect the whole soil quality under various vegetation types. Overall, this study might identify existing health problems and provide insights for enhancing protection and utilization management for mangrove ecosystem in the Greater Bay Area.

Development and validation of novel 8-dye short tandem repeat multiplex system for forensic applications.

DNA profiling of short tandem repeats (STRs) is the primary method for genotyping forensic samples. However, degraded DNA and trace samples are still major problems for commercial 5- or 6-dye STR kits. In order to improve the performance of this method, we developed a novel 8-dye STR multiplex system containing 18 autosomal loci (D3S1358, D1S1656, TPOX, D16S539, vWA, D6S1043, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, TH01, D21S11, D12S391, and PentaD) and the sex-determining locus Amelogenin, with all fragments smaller than 330 bases. Validation was carried out as recommended by the Scientific Working Group on DNA Analysis Methods. The results showed that complete profiles were obtainable when the input DNA was as low as 0.0625 ng. Full profiles were obtained even in the presence of inhibitors such as humic acid (< 300 ng/µl), hematin (< 100 µM), and indigo (0.01%). The 8-dye STR multiplex system also showed good performance in the detection degraded DNA samples. These results indicate that the 8-dye STR multiplex system is suitable for human DNA genotyping, including for difficult forensic materials.

Considering the flanking region variants of nonbinary SNP and phenotype-informative SNP to constitute 30 microhaplotype loci for increasing the discriminative ability of forensic applications.

The flanking region variants of nonbinary SNPs and phenotype-informative SNPs (piSNPs) have been observed, which may greatly improve the discriminative ability after constituting microhaplotype. In this study, 30 microhaplotype loci based on the nonbinary SNPs and piSNPs (shown to be related to phenotypes such as hair and eye color) were selected. Genotyping were conducted on 100 unrelated northern Han Chinese, and the 26 populations from the 1000 Genome Project were also included for comparison of populations differentiation. The simulated study was conducted for evaluating the efficiency of kinship testing. These 30 microhaplotype loci we selected had good polymorphism, with a mean effective number of alleles (Ae) of 3.46. The average Ae increase was 1.27 compared with the target SNPs. The populations from the five regions worldwide could also be distinguished using these loci. The results of kinship testing showed that these microhaplotype loci had the similar ability as 15 STR loci of AmpFlSTRR IdentifilerR PCR Amplification Kit to identify the biological parent and a stronger ability to exclude the nonbiological parents. So, these 30 microhaplotype loci may be multifunctional for forensic application, including the ability of personal identification and kinship testing equivalent to 15 STR loci, and the power of ancestry inference for distinguishing the main intercontinental population. Moreover, our selected phenotypic microhaplotype loci may theoretically have phenotype prediction capabilities. But the phenotype prediction efficiency of these phenotypic microhaplotype loci may be worse than that of piSNPs and the detailed prediction accuracy of different populations needs to be further studied.

Developmental validation of the STRscan-17LC kit: a 6 Dye STR kit enhanced stability and ability to detect degraded samples.

Genotyping of short tandem repeat (STR) markers is the basic method of forensic science. Enhanced technologies are needed to meet the requirements of databasing and casework samples. The STRscan-17LC kit is a 6 Dye STR kit which amplifies 16 STR loci: D3S1358, TPOX, D16S539, vWA, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, D18S51, TH01, D12S391, and D21S11 and the sex-determinant locus amelogenin. This kit is designed for better tolerance to PCR inhibitors and analysis of mildly degraded samples with all fragments smaller than 330 bases. In this study, the STRscan-17LC kit is validated according to the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines, including PCR-based studies, sensitivity, precision and accuracy, inhibitors, species specificity, DNA mixture studies, population, and concordance studies. The validation results suggest that the STRscan-17LC kit is a useful tool for forensic application.

An 18 Multi-InDels panel for analysis of highly degraded forensic biological samples.

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10-12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.

Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis.

Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB-sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.

Construction of a library of cloned short tandem repeat (STR) alleles as universal templates for allelic ladder preparation.

Short tandem repeat (STR) genotyping methods are widely used for human identity testing applications, including forensic DNA analysis. Samples of DNA containing the length-variant STR alleles are typically separated and genotyped by comparison to an allelic ladder. Here, we describe a newly devised library of cloned STR alleles. The library covers alleles X and Y for the sex-determining locus Amelogenin and 259 other alleles for 22 autosomal STR loci (TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D21S11, D2S1338, D6S1043, D12S391, Penta E, D19S433, D11S4463, D17S974, D3S4529 and D12ATA63). New primers were designed for all these loci to construct recombinant plasmids so that the library retains core repeat elements of STR as well as 5'- and 3'-flanking sequences of ∼500 base pairs. Since amplicons of commercial STR genotyping kits and systems developed in laboratories are usually distributed from 50 to <500 base pairs, this library could provide universal templates for allelic ladder preparation. We prepared three different sets of allelic ladders for this locus TH01 and an updated version of an allelic ladder for the DNATyper(®)19 multiplex system using these plasmids to confirm the suitability of the library as a good source for allelic ladder preparation. Importantly, the authenticity of each construct was confirmed by bidirectional nucleotide sequencing and we report the repeat structures of the 259 STR alleles. The sequencing results showed all repeat structures we obtained for TPOX, CSF1PO, D7S820, TH01, D16S539, D18S51 and Penta E were the same as reported. However, we identified 102 unreported repeat structures from the other 15 STR loci, supplementing our current knowledge of repeat structures and leading to further understanding of these widely used loci.

Nanoplasmonic imaging of latent fingerprints and identification of cocaine.

Search for traces: Aptamer-bound Au nanoparticles (Au NPs) were used to provide high-resolution dark-field microscopy images of latent fingerprints (LFPs) with level 2 and level 3 details. Furthermore, the cocaine-induced aggregation of Au NPs results in a true green-to-red color change of the scattered light, providing a quasi-quantative method to identify cocaine loadings in LFPs.

A power-free microfluidic chip for SNP genotyping using graphene oxide and a DNA intercalating dye.

We herein report a power-free microfluidic chip for fluorescent DNA detection with high single-nucleotide polymorphism discrimination, using a DNA intercalator and graphene oxide.

A graphene-based platform for fluorescent detection of SNPs.

A novel fluorescent single nucleotide polymorphism (SNP) assay was developed by using Graphene Oxide (GO), which provides a fast, sensitive and simple method for SNP detection. The strategy was based on the single base extension reaction and different absorption capacity of fluorescein labeled dGTP (dGTP-Fl) and double-stranded DNA (dsDNA) to GO. dGTP-Fl is incorporated into the probe by extension reaction for the mutant target but not for the wild target, which leads to recovered fluorescence for the mutant target because of weak interaction between dsDNA and GO and weak fluorescence for the wild target because of the quenched fluorescence of dGTP-Fl by GO. The method shows a linear range for the mutant-type target from 3 nM to 50 nM and 3 nM is the detection limit. It was noted that as low as 10% mutant-type target could be detected in the presence of the wild-type target, in which the concentration is 9 times higher than that of the mutant-type target.