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.

Validation of the PowerPlex®35GY System: a novel eight-dye STR multiplex kit on the Spectrum Compact CE System.

The PowerPlex® 35GY System (Promega, USA) is an advanced eight-dye multiplex STR kit, incorporating twenty-three autosomal STR loci, eleven Y chromosome STR loci, one sex determining marker Amelogenin, and two quality indicators. This multiplex system includes 20 CODIS loci and up to 15 mini-STR loci with sizing values less than 250 bases. In this study, validation for PowerPlex® 35GY System was conducted following the guidelines of SWGDAM, encompassing sensitivity, precision, accuracy, concordance, species specificity, stutter, mixture, stability, and degraded DNA. The results from experiments demonstrated that the PowerPlex® 35GY System could effectively amplify DNA samples, with complete allele detection achieved at 125 pg. Moreover, over 90% of alleles from minor contributors were detected at a mixed ratio of 1:4. Additionally, the system was found to yield full profiles even in the presence of hematin, humic acid, and indigo. The PowerPlex® 35GY System demonstrated superior performance in the sensitivity and degraded DNA studies compared to a six-dye STR kit. Hence, it is evident that the PowerPlex® 35GY System is well-suited for forensic practice, whether in casework or for database samples. These findings provide strong support for the efficacy and reliability of the PowerPlex® 35GY System in forensic applications.

Value of ultrathin bronchoscope in improving the endobronchial ultrasound localization rate and diagnosing peripheral pulmonary nodules by cryobiopsy.

BACKGROUND: A 3.0-mm ultrathin bronchoscope (UTB) with a 1.7-mm working channel provides better accessibility to peripheral bronchi. A 4.0-mm thin bronchoscope with a larger 2.0-mm working channel facilitates the use of a guide sheath (GS), ensuring repeated sampling from the same location. The 1.1-mm ultrathin cryoprobe has a smaller diameter, overcoming the limitation of the size of biopsy instruments used with UTB. In this study, we compared the endobronchial ultrasound localization rate and diagnostic yield of peripheral lung lesions by cryobiopsy using UTB and thin bronchoscopy combined with GS. METHODS: We retrospectively evaluated 133 patients with peripheral pulmonary lesions with a diameter less than 30 mm who underwent bronchoscopy with either thin bronchoscope or UTB from May 2019 to May 2023. A 3.0-mm UTB combined with rEBUS was used in the UTB group, whereas a 4.0-mm thin bronchoscope combined with rEBUS and GS was used for the thin bronchoscope group. A 1.1-mm ultrathin cryoprobe was used for cryobiopsy in the two groups. RESULTS: Among the 133 patients, peripheral pulmonary nodules in 85 subjects were visualized using r-EBUS. The ultrasound localization rate was significantly higher in the UTB group than in the thin bronchoscope group (96.0% vs. 44.6%, respectively; P < 0.001). The diagnostic yield of cryobiopsy specimens from the UTB group was significantly higher compared to the thin bronchoscope group (54.0% vs. 30.1%, respectively; p = 0.006). Univariate analysis demonstrated that the cryobiopsy diagnostic yields of the UTB group were significantly higher for lesions ≤ 20 mm, benign lesions, upper lobe lesions, lesions located lateral one-third from the hilum, and lesions without bronchus sign. CONCLUSIONS: Ultrathin bronchoscopy combined with cryobiopsy has a superior ultrasound localization rate and diagnostic yield compared to a combination of cryobiopsy and thin bronchoscopy.

Interleukin-32γ promotes macrophage-mediated chemoresistance by inducing CSF1-dependent M2 macrophage polarization in multiple myeloma.

Macrophages (MΦs) are an abundant component in the multiple myeloma (MM) environment and contribute to MM drug resistance. We previously showed that interleukin-32 (IL-32) is highly expressed in MM patients and induces the immunosuppressive function of MΦs. The present study was designed to explore the role of IL-32 in MΦ-mediated MM drug resistance and the underlying mechanism. Our analysis revealed that IL-32 expression was upregulated in relapsed MM patients and associated with CD206+ M2 MΦ infiltration. Subsequently, we found that the most active isoform, IL-32γ, promoted MΦs to protect MM cells from drug-induced apoptosis both in vitro and in vivo. Furthermore, by evaluating many parameters, including surface markers, cytokines, metabolic enzymes and characteristic molecules, IL-32γ was verified to induce the polarization of M2 MΦs, a function that was partly dependent on increasing the expression of colony-stimulating factor 1 (CSF1). Taken together, the results of our study indicate that IL-32γ promotes MΦ-mediated MM drug resistance and modifies MΦs toward the M2 phenotype, providing a crucial theoretical basis for targeted MΦ immunotherapy.

The NS4A Protein of Classical Swine Fever Virus Suppresses RNA Silencing in Mammalian Cells.

RNA interference (RNAi) is a significant posttranscriptional gene silencing mechanism and can function as an antiviral immunity in eukaryotes. However, numerous viruses can evade this antiviral RNAi by encoding viral suppressors of RNA silencing (VSRs). Classical swine fever virus (CSFV), belonging to the genus Pestivirus, is the cause of classical swine fever (CSF), which has an enormous impact on animal health and the pig industry. Notably, little is known about how Pestivirus blocks RNAi in their host. In this paper, we uncovered that CSFV NS4A protein can antagonize RNAi efficiently in mammalian cells by binding to double-stranded RNA and small interfering RNA. In addition, the VSR activity of CSFV NS4A was conserved among Pestivirus. Furthermore, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi in mammalian cells. In conclusion, our studies uncovered that CSFV NS4A is a novel VSR that suppresses RNAi in mammalian cells and shed new light on knowledge about CSFV and other Pestivirus. IMPORTANCE It is well known that RNAi is an important posttranscriptional gene silencing mechanism that is also involved in the antiviral response in mammalian cells. While numerous viruses have evolved to block this antiviral immunity by encoding VSRs. Our data demonstrated that the NS4A protein of CSFV exhibited a potent VSR activity through binding to dsRNA and siRNA in the context of CSFV infection in mammalian cells, which are a conservative feature among Pestivirus. In addition, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi, providing a theoretical basis for the development of other important attenuated Pestivirus vaccines.

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.

Preliminary study on genetic factors related to Demirjian's tooth age estimation method based on genome-wide association analysis.

The age determination of individuals, especially minors, is critical in forensic research. In forensic practice, dental age estimation is one of the most commonly used methods for determining age as teeth are easy to preserve and relatively resistant to environmental factors. Tooth development is affected and regulated by genetic factors; however, these are not incorporated into current commonly used tooth age inference methods, leading to unreliable results. Here, we established a Demirjian and a Cameriere tooth age estimation-based methods suitable for use in children in southern China. By using the difference between the inferred age and the actual age (MD) as the phenotype, we identified 65 and 49 SNPs related to tooth age estimation from 743,722 loci among 171 children in southern China through a genome-wide association analysis (p<0.0001). We also conducted a genome-wide association study on dental development stage (DD) using the Demirjian tooth age estimation method and screened two sets of SNP sites (52 and 26) based on whether age difference was considered. The gene function enrichment analysis of these SNPs found that they were related to bone development and mineralization. Although SNP sites screened based on MD seem to improve the accuracy of tooth age estimation, there is little correlation between these SNPs and an individual's Demirjian morphological stage. In conclusion, we found that individual genotypes can affect tooth age estimation, and based on different phenotypic analysis models, we have identified some novel SNP sites related to tooth age inference and Demirjian's tooth development stage. These studies provide a reference for subsequent phenotypic selection based on tooth age inference analysis, and the results could possibly be used in the future to make forensic age estimation more accurate.

Warming Menstruation and Analgesic Soup Inhibits PD-1/PD-L1 Pathway in Rats with Endometriosis.

Aim: To observe the effect of warming menstruation and analgesic herbal soup (WMAS) on the pathway of programmed cell death protein 1 and its ligand 1 PD-1/PD-L1 in rats with endometriosis model. Methods: A total of 90 mature female Wistar rats were randomly divided into 6 groups of 15 rats each. Of these, 5 groups were randomly selected for endometriosis molding and given high (HW group), medium (MW group) and low (LW group) doses of WMAS, western medicine (progesterone capsules, PC group) and saline gavage (SG group) respectively. The other group was a normal group (NM group), which was given saline gavage. The protein expression of PD-1 and PD-L1 on rat in eutopic and ectopic endothelium was detected by immunohistochemistry and the mRNA expression of PD-1 and PD-L1 in rats was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). Results: The protein and mRNA expression of PD-1 and PD-L in the eutopic and ectopic endometrium of rats in the endometriosis group were higher than in the normal group (P <.05). The protein and mRNA expression of PD-1 and PD-L1 in the eutopic and ectopic endothelium of the HW, MW and PC groups were lower than in the SG group (P <.05). Conclusion: High expression of PD-1 and PD-L1 occurs in endometriosis, and WMAS can inhibit the immune signalling pathway PD-1/PD-L1, which may be available to inhibit the development of endometriosis.

Application of Microhaplotypes in Sibling Kinship Testing.

OBJECTIVES: To investigate the efficacy of different numbers of microhaplotype (MH) loci and the introduction of different reference samples on the identification of full sibling, half sibling and differentiation between full sibling and half sibling kinships, and to explore the effect of changing mutation rate on sibling testing. METHODS: First, a family map involving three generations was established, and four full sibling identification models, five half sibling identification models and five models distinguishing full and half siblings were constructed for different reference samples introduced. Based on the results of the previous study, two sets of nonbinary SNP-MH containing 34 and 54 loci were selected. Based on the above MH loci, 100 000 pairs of full sibling vs. unrelated individuals, 100 000 pairs of half sibling vs. unrelated individuals and 100 000 pairs of full sibling vs. half sibling were simulated based on the corresponding sibling kinship testing models, and the efficacy of each sibling kinship testing model was analyzed by the likelihood ratio algorithm under different thresholds. The mutant rate of 54 MH loci was changed to analyze the effect of mutation rate on sibling identification. RESULTS: In the same relationship testing model, the systematic efficacy of sibling testing was positively correlated with the number of MH loci detected. With the same number of MH loci, the efficacy of full sibling testing was better than that of uncle or grandfather when the reference sample introduced was a full sibling of A, but there was no significant difference in the identification efficacy of the four reference samples introduced for full sibling and half sibling differentiation testing. In addition, the mutation rate had a slight effect on the efficacy of sibling kinship testing. CONCLUSIONS: Increasing the number of MH loci and introducing reference samples of known relatives can increase the efficacy of full sibling testing, half sibling testing, and differentiation between full and half sibling kinships. The level of mutation rate in sibling testing by likelihood ratio method has a slight but insignificant effect on the efficacy.

Near-Infrared Dye-Aptamer Assay for Small Molecule Detection in Complex Specimens.

Aptamers are single-stranded oligonucleotides isolated in vitro that bind specific targets with high affinity and are commonly used as receptors in biosensors. Aptamer-based dye-displacement assays are a promising sensing platform because they are label-free, sensitive, simple, and rapid. However, these assays can exhibit impaired sensitivity in biospecimens, which contain numerous interferents that cause unwanted absorbance, scattering, and fluorescence in the UV-vis region. Here, this problem is overcome by utilizing near-infrared (NIR) signatures of the dye 3,3'-diethylthiadicarbocyanine iodide (Cy5). Cy5 initially complexes with aptamers as monomers and dimers; aptamer-target binding displaces the dye into solution, resulting in the formation of J-aggregates that provide a detectable NIR signal. The generality of our assay is demonstrated by detecting three different small-molecule analytes with their respective DNA aptamers at clinically relevant concentrations in serum and urine. These successful demonstrations show the utility of dye-aptamer NIR biosensors for high-throughput detection of analytes in clinical specimens.

Detection and quantification of glycosylated queuosine modified tRNAs by acid denaturing and APB gels.

Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G34U35N36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNATyr and tRNAAsp are further glycosylated with galactose and mannose, respectively. Although galactosyl-Q (galQ) and mannosyl-Q (manQ) can be measured by LC/MS approaches, the difficulty of detecting and quantifying these modifications with low sample inputs has hindered their biological investigations. Here we describe a simple acid denaturing gel and nonradioactive northern blot method to detect and quantify the fraction of galQ/manQ-modified tRNA using just microgram amounts of total RNA. Our method relies on the secondary amine group of galQ/manQ becoming positively charged to slow their migration in acid denaturing gels commonly used for tRNA charging studies. We apply this method to determine the Q and galQ/manQ modification kinetics in three human cells lines. For Q-modification, tRNAAsp is modified the fastest, followed by tRNAHis, tRNATyr, and tRNAAsn Compared to Q-modification, glycosylation occurs at a much slower rate for tRNAAsp, but at a similar rate for tRNATyr Our method enables easy access to study the function of these enigmatic tRNA modifications.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA.IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.

Study on the biogas potential of anaerobic digestion of coffee husks wastes in Ethiopia.

The poorly controlled discharge of coffee husks in Ethiopia causes severe environmental pollution and is a waste of resources. The volatile solid and carbon content in coffee husks waste indicates that it is rich in organic matter and has huge potential to produce biogas. This study investigated the feasibility of coffee husks to produce biomass through anaerobic digestion, based on temperature, initial pH, inoculum/substrate (I/S) ratio and carbon/nitrogen (C/N) ratio. The study demonstrated that the maximum production of biogas and methane reached 3359.6 ml and 2127.30 ml, respectively, under the conditions of mesophilic temperature (35±1°C), an initial pH of 7, an I/S ratio of 0.75 and a C/N ratio of 30. Based on this result, the effects of trace elements (Fe2+, Ni2+, Co2+) on biogas production and methane content were also explored. Compared with the group with no addition of trace elements, the experiment adding trace elements had significant enhancement effects on the production of biogas and methane, in which Fe2+ played a leading role (p<0.05). Fe2+ promoted the hydrolysis and acidification of coffee husks, resulting in the production of a series of intermediates such as volatile fatty acids and the other kinds of dissolved organic matter. Furthermore, the cooperation of Ni2+, Co2+ and Fe2+ enhanced the activity of the enzyme system in methanogens, promoting methane production. The results in this paper show that coffee husks have clear biogas potential through anaerobic digestion, and its effective utilization could fulfill the dual purpose of solid waste reclamation and local environmental protection in Ethiopia.

Association Between Age and the 28-Day All-Cause Mortality in Tuberculosis Complicated by Sepsis in ICU Patients: A Retrospective Cohort Study.

Purpose: Age is considered a vital factor in intensive care units (ICUs) because of its association with physiological frailty, comorbidities, and immune system function. Previous studies have examined the association between age and prognosis in patients with tuberculosis (TB) or sepsis; however, the association between age and prognosis in ICU patients with TB complicated by sepsis is rare. This study aimed to assess the association between age and the prognosis of patients in the ICU with TB complicated by sepsis. Patients and Methods: Data from the ICU of the Public Health Clinical Center of Chengdu were analyzed using the multivariable Cox regression model, stratified analysis with interaction, restricted cubic spline (RCS), and threshold effect analysis to investigate the association between age and 28-day all-cause mortality in patients with TB complicated by sepsis. Results: In total, 520 patients diagnosed with TB and sepsis were enrolled (120 women [23.1%]; median age, 64 years). The association between age and risk of death exhibited a J-shaped curve on the RCS (P for nonlinearity = 0.001). In the threshold analysis, the hazard ratio for the risk of death was 1.104 (95% confidence interval, 1.05-1.16) in participants aged ≥66.2 years. The risk of death increased by 10.4% with every 1-year increase in age in patients with TB complicated by sepsis. No significant association was found between age and 28-day all-cause mortality in patients aged <66.2 years. Conclusion: A nonlinear relationship was observed between age and short-term all-cause mortality in patients in the ICU with TB complicated by sepsis. Patients with a higher age at admission may have a higher risk of death and require focused attention, close monitoring, and early treatment to reduce mortality.

The association between blood urea nitrogen to albumin ratio and the 28 day mortality in tuberculosis patients complicated by sepsis.

The relationship between blood urea nitrogen to albumin ratio (BAR) and the prognosis of patients with tuberculosis (TB) complicated by sepsis remains unclear. This study aimed to explore the association between BAR and overall patient prognosis. This was a retrospective cohort study of patients with TB complicated by sepsis who were admitted to the intensive care unit (ICU) of the Public Health Clinical Center of Chengdu between January 2019 and February 2023. The relationship between BAR values and prognosis in these patients was investigated using multivariate Cox regression, stratified analysis with interaction, restricted cubic spline (RCS), and threshold effect analysis. Sensitivity analyses were conducted to assess the robustness of the results. Our study included 537 TB patients complicated by sepsis admitted in the ICU, with a median age of 63.0 (48.0, 72.0) years; 76.7% of whom were men. The multivariate-restricted cubic spline analysis showed a non-linear association between BAR and patient prognosis. In the threshold analysis, we found that TB patients complicated by sepsis and a BAR < 7.916 mg/g had an adjusted hazard ratio (HR) for prognosis of 1.163 (95% CI 1.038-1.303; P = 0.009). However, when the BAR was ≥ 7.916 mg/g, there was no significant increase in the risk of death. The results of the sensitivity analysis were stable.

Flow-induced fabrication of ZnO nanostructures in pillar-arrayed microchannels.

The emergence of microfluidic devices integrated with nanostructures enables highly efficient, flexible and controllable biosensing, among which zinc oxide (ZnO) nanostructure-based fluorescence detection has been demonstrated to be a promising methodology due to its high electrical point and unique fluorescence enhancement properties. The optimization of microfluidic synthesis of ZnO nanostructures for biosensing on chip has been in demand due to its low cost and high efficiency, but still the flow-induced growth of ZnO nanostructures is not extensively studied. Here, we report a simple and versatile strategy that could manipulate the local flow field by creating periodically arranged micropillars within a straight microchannel. We have explored the effects of perfusion speed and flow direction of seed solution, localized flow variation of growth solution and growth time on the morphology of nanostructures. This provided a comprehensive understanding which governs nanostructure fabrication controlled by flow. The results demonstrated that localized flow in microfluidic devices was essential for the initiation and growth of zinc oxide crystals, enabling precise control over their properties and morphology. Furthermore, a model protein was used to demonstrate the intrinsic fluorescence enhancement of ZnO nanostructures as an example to reveal the morphology-related enhancement properties.

SDH, a novel diarylheptane compound, alleviates dextran sulfate sodium (DSS)-induced colitis by reducing Th1/Th2/Th17 induction and regulating the gut microbiota in mice.

Ulcerative colitis, a chronic inflammatory condition affecting the rectum and colon to varying degrees, is linked to a dysregulated immune response and the microbiota. Sodium (aS,9R)-3-hydroxy-16,17-dimethoxy-15-oxidotricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexene-9-yl sulfate hydrate (SDH) emerges as a novel diarylheptane compound aimed at treating inflammatory bowel diseases. However, the mechanisms by which SDH modulates these conditions remain largely unknown. In this study, we assessed SDH's impact on the clinical progression of dextran sodium sulfate (DSS)-induced ulcerative colitis. Our results demonstrated that SDH significantly mitigated the symptoms of DSS-induced colitis, reflected in reduced disease activity index scores, alleviation of weight loss, shortening of the colorectum, and reduction in spleen swelling. Notably, SDH decreased the proportion of Th1/Th2/Th17 cells and normalized inflammatory cytokine levels in the colon. Furthermore, SDH treatment modified the gut microbial composition in mice with colitis, notably decreasing Bacteroidetes and Proteobacteria populations while substantially increasing Firmicutes, Actinobacteria, and Patescibacteria. In conclusion, our findings suggest that SDH may protect the colon from DSS-induced colitis through the regulation of Th1/Th2/Th17 cells and gut microbiota, offering novel insights into SDH's therapeutic potential.

Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.

TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

A preliminary study on identification of the blood donor in a body fluid mixture using a novel compound genetic marker blood-specific methylation-microhaplotype.

Blood-containing mixtures are frequently encountered at crime scenes involving violence and murder. However, the presence of blood, and the association of blood with a specific donor within these mixtures present significant challenges in forensic analysis. In light of these challenges, this study sought to address these issues by leveraging blood-specific methylation sites and closely linked microhaplotype sites, proposing a novel composite genetic marker known as "blood-specific methylation-microhaplotype". This marker was designed to the detection of blood and the determination of blood donor within blood-containing mixtures. According to the selection criteria mentioned in the Materials and Methods section, we selected 10 blood-specific methylation-microhaplotype loci for inclusion in this study. Among these loci, eight exhibited blood-specific hypomethylation, while the remaining two displayed blood-specific hypermethylation. Based on data obtained from 124 individual samples in our study, the combined discrimination power (CPD) of these 10 successfully sequenced loci was 0.999999298. The sample allele methylation rate (Ram) was obtained from massive parallel sequencing (MPS), which was defined as the proportion of methylated reads to the total clustered reads that were genotyped to a specific allele. To develop an allele type classification model capable of identifying the presence of blood and the blood donor, we used the Random Forest algorithm. This model was trained and evaluated using the Ram distribution of individual samples and the Ram distribution of simulated shared alleles. Subsequently, we applied the developed allele type classification model to predict alleles within actual mixtures, trying to exclude non-blood-specific alleles, ultimately allowing us to identify the presence of blood and the blood donor in the blood-containing mixtures. Our findings demonstrate that these blood-specific methylation-microhaplotype loci have the capability to not only detect the presence of blood but also accurately associate blood with the true donor in blood-containing mixtures with the mixing ratios of 1:29, 1:19, 1:9, 1:4, 1:2, 2:1, 7:1, 8:1, 31:1 and 36:1 (blood:non-blood) by DNA mixture interpretation methods. In addition, the presence of blood and the true blood donor could be identified in a mixture containing four body fluids (blood:vaginal fluid:semen:saliva = 1:1:1:1). It is important to note that while these loci exhibit great potential, the impact of allele dropouts and alleles misidentification must be considered when interpreting the results. This is a preliminary study utilising blood-specific methylation-microhaplotype as a complementary tool to other well-established genetic markers (STR, SNP, microhaplotype, etc.) for the analysis in blood-containing mixtures.