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.

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.

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.

Association of Depression with Long-Term Cardiovascular Risks in Older Patients with Obstructive Sleep Apnea.

Introduction: Obstructive sleep apnea (OSA) is associated with an increased risk of cardiovascular disease (CVD). Depression is a crucial factor among the various factors that are associated with OSA and CVD. Purpose: This study was conducted with an aim to assess the prognostic significance of depression on the MACE in older patients with OSA. Patients and Methods: 1106 older patients with OSA, without myocardial infarction (MI), history of hospitalization for unstable angina, or heart failure at baseline were enrolled and followed up prospectively. Incidence rates were expressed as cumulative incidence. Cox proportional hazards analysis was used to estimate the risk of all events. The primary outcomes were major adverse cardiovascular events (MACE). Each patient underwent polysomnography (PSG) and GDS-12 scale assessment. Those with an apnea-hypopnea index (AHI) greater than 5 were diagnosed with OSA, while those with a scale score greater than 3 were diagnosed with depression. Results: Among the 1106 older patients with OSA, depression was found in 133(12.0%) patients, 96(8.7%) patients experienced MACE during the follow-up. Depression was associated with a higher cumulative incidence of MACE in older patients with OSA. Multivariate analysis revealed that depression independently increased the risk of MACE (adjusted hazard ratio [aHR] = 2.29; 95% confidence interval [CI]: 1.34-3.90; P = 0.002). Subgroup analyses showed that male patients (aHR = 2.96; 95% CI: 1.52-5.77; P = 0.001), overweight-obese individuals (aHR = 2.98; 95% CI: 1.49-6.00; P = 0.002), and those with moderate-severe OSA (aHR = 2.82; 95% CI: 1.55-5.14; P = 0.001) and concurrent depression were at a higher risk for MACE. Conclusion: Depression is common in older patients with OSA in the absence of MI, hospitalization for unstable angina, or heart failure, and confers an independent, increased risk of MACE.

Evaluating the effect of Roxadustat on ventricular repolarization in patients undergoing peritoneal dialysis.

BACKGROUND: Roxadustat is a novel oral medication used to treat anemia in CKD patients. Several studies have shown that Roxadustat can alleviate anemia in CKD patients by increasing hemoglobin levels and regulating iron metabolism. We aimed to evaluate the effect of Roxadustat on ventricular repolarization in PD patients. This study may provide a new integrated approach to the assessment and treatment of CKD. METHODS: The present prospective cohort study enrolled 65 CKD patients who were treated with Roxadustat and 31 CKD patients who received conventional therapy between January 2021 and June 2022. All patients were examined for ECG in the absence of clinical symptoms and compared the ECG indicators. Demographic and clinical data of all patients were collected. All data used SPSS 18.0 for statistical analyses. RESULTS: The T peak-to-end (Tpe) of PD patients in the Roxadustat group was remarkably slower than that of patients in the conventional group. Additionally, the Tpe/QT ratio in the conventional group was significantly elevated than that in the Roxadustat group. The results of logistic regression analysis showed that Tpe (95%CI 1.191 ~ 2.141, P = 0.002) and Roxadustat treatment (95%CI 1.357 ~ 42.121, P = 0.021) were the risk factors of PD patients with high Tp-e/QT ratio. CONCLUSION: In summary, we found that Roxadustat could improve ventricular repolarization in peritoneal dialysis patients, which indicated a potential cardiovascular protective effect of Roxadustat. This study might provide a new integrated approach to the assessment and treatment of CKD.

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.

A Prospective Case-Control Study Examining the Relationship Between Frailty and Serum Myostatin in Older Persons with Chronic Heart Failure.

Background: Frailty affects the prognosis and management of patients with heart failure, and is often related with sarcopenia. Also, the serum myostatin (MSTN) involved in the development of sarcopenia and frailty. This study aimed to determine the connection between MSTN level and frailty in older adults with chronic heart failure (CHF). Methods: This prospective case-control study enrolled older adult patients with CHF between May 2019 and May 2021, and analyzed their clinical data. Results: In this study 75 older adults with CHF were included, 29 of whom were frail. The B-type natriuretic peptide (BNP) levels were significantly higher in frail older adults with CHF than in older adults with CHF who were not frail (316.82 ± 235.64 pg/mL vs 198.61 ± 112.58 pg/mL; P = 0.016). The MSTN levels were significantly higher in frail participants than in participants who were not frail (2.93 ± 1.35 ng/mL vs 2.24 ± 0.84 ng/mL; P = 0.018). Based on multivariable analysis the BNP (odds ratio [OR] = 1.004, 95% confidence interval [CI] = 1 0.001-1.008; P = 0.018) and MSTN (OR = 1.772, 95% CI = 1.079-2.912; P =0 0.024) levels were independently associated with frailty in older adults with CHF. Conclusion: MSTN is a promising biomarker of frailty in elderly patients with CHF.

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.

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.

High-resolution structural study on pyridin-3-yl ebselen and its N-methylated tosylate and iodide derivatives.

The crystal structure of the pyridine-substituted benzisoselenazolinone 2-(pyridin-3-yl)-2,3-dihydro-1,2-benzoselenazol-3-one (C12H8N2OSe, 2), related to the antioxidant ebselen [systematic name: 2-phenyl-1,2-benzoselenazol-3(2H)-one, 1], is characterized by strong intermolecular N...Se(-N) chalcogen bonding, where the N...Se distance of 2.3831 (6) Šis well within the sum of the van der Waals radii for N and Se (3.34 Å). This strong interaction results in significant lengthening of the internal N-Se distance, consistent with significant population of the Se-N σ* antibonding orbital. Much weaker intermolecular O...Se chalcogen bonding occurs between the amide-like O atom in 2 and the less polarized C-Se bond in this structure. Charge density analysis of 2 using multipole refinement of high-resolution data allowed the electrostatic surface potential for 2 to be mapped, and clearly reveals the σ-hole at the extension of the Se-N bond as an area of positive electrostatic potential. Topological analysis of the electron-density distribution in 2 was carried out within the Quantum Theory of Atoms in Molecules (QTAIM) framework and revealed bond paths and (3,-1) bond critical points (BCPs) for the N...Se-N moiety consistent with a closed-shell interaction; however, the potential energy term is suggestive of electron sharing. Analysis of the electron localization function (ELF) for the strong N...Se and the weak O...Se chalcogen-bonding interactions in the structure of 2 suggest significant electron sharing in the former interaction, and a largely electrostatic interaction in the latter. Conversion of 2 to its N-methylated derivatives by reaction with methyl iodide [1-methyl-3-(3-oxo-2,3-dihydro-1,2-benzoselenazol-2-yl)pyridin-1-ium iodide, C13H11N2OSe+·I-] and methyl tosylate [1-methyl-3-(3-oxo-2,3-dihydro-1,2-benzoselenazol-2-yl)pyridin-1-ium toluenesulfonate trihydrate, C13H11N2OSe+·C7H7O3S-·3H2O] removes the possibility of N...Se chalcogen bonding and instead structures are obtained where the iodide and tosylate counter-ions fulfill the role of chalcogen-bond acceptors, with a strong I-...Se interaction in the iodide salt and a weaker p-Tol-SO3-...Se interaction in the tosylate salt.

Bergenin alleviates Diabetic Retinopathy in STZ-induced rats.

Diabetic retinopathy (DR) is the key cause of blindness and visual impairment in diabetes patients around the world. The high levels of oxidative stress in diabetes patients cause diabetic retinopathy. In addition to being an antioxidant, Bergenin also works as an immunosuppressant, an anti-inflammatory, and anticarcinogenic against hepatocarcinoma. This study examined the effects of Bergenin on diabetic retinopathy rats, using Streptozotocin (STZ) intraperitoneally to induce diabetes in rats. The animals were divided into four groups (n = 6), including a normal control (Group I), diabetic control (Group II), Bergenin (25 mg/kg) (Group III), and metformin (350 mg/kg) (Group IV). As previously mentioned, each animal received treatment for 60 days. To induce DR, rats were administered STZ (60 mg/kg) intraperitoneally for 60 days. Standard methods were utilized to measure the body weight of rats, blood glucose levels. We measured lipid profiles (Triglycerides, cholesterol, LDL, and HDL), inflammatory markers, and antioxidant levels with their respective kits. Analysis of retinal tissue morphometry and MMP-9, VEGF, and MCP-1 levels in serum was performed. Our research examined the expression levels of target genes (TNF-α, IL-1ß, and IL-6) using RT-PCR analysis. STZ-induced animals that were treated with Bergenin had less food intake, lower blood glucose, and improved body weight. Bergenin significantly suppressed levels of pro-inflammatory cytokines, cholesterol, TG, LDL, AI, MMP-9, VEGF, and MCP-1 and increased the level of HDL and antioxidant enzymes in STZ-induced DR rats. As well as increasing antioxidant levels, reducing retinal thickness, and increasing cell numbers, Bergenin also lessened DR remarkably. The results of this study demonstrated that Bergenin effectively inhibited STZ-induced DR in rats.

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.

Advice for smokers in smoking cessation clinic: a review.

Background: Tobacco dependence has become a global public health concern. We chose to investigate the modifiable factors and motivations during the period of smoking cessation based on the mechanism of nicotine addiction. Methods: We selected emotion, sleep, alcohol, caffeine beverages, mental activities after dinner, exercise and CYP2A6 genotype as influencing factors, and provided corresponding recommendations for smokers based on these factors. Based on these characteristics, we reviewed literature and summarized the relationship between these factors and nicotine dependence or smoking. Results: Different emotion, sleep deficiency, caffeine intake, alcohol consumption, mental activities after dinner, physical exercises and CYP2A6 genotype have an effect on daily smoking and nicotine dependence. Conclusion: These suggestions related literature-derived factors may increase the success rate of smoking cessation.

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.

The application of short and highly polymorphic microhaplotype loci in paternity testing and sibling testing of temperature-dependent degraded samples.

Paternity testing and sibling testing become more complex and difficult when samples degrade. But the commonly used genetic markers (STR and SNP) cannot completely solve this problem due to some disadvantages. The novel genetic marker microhaplotype proposed by Kidd's research group combines the advantages of STR and SNP and is expected to become a promising genetic marker for kinship testing in degraded samples. Therefore, in this study, we intended to select an appropriate number of highly polymorphic SNP-based microhaplotype loci, detect them by the next-generation sequencing technology, analyze their ability to detect degraded samples, calculate their forensic parameters based on the collected 96 unrelated individuals, and evaluate their effectiveness in paternity testing and sibling testing by simulating kinship relationship pairs, which were also compared to 15 STR loci. Finally, a short and highly polymorphic microhaplotype panel was developed, containing 36 highly polymorphic SNP-based microhaplotype loci with lengths smaller than 100 bp and A e greater than 3.00, of which 29 microhaplotype loci could not reject the Hardy-Weinberg equilibrium and linkage equilibrium after the Bonferroni correction. The CPD and CPE of these 29 microhaplotype loci were 1-2.96E-26 and 1-5.45E-09, respectively. No allele dropout was observed in degraded samples incubated with 100°C hot water for 40min and 60min. According to the simulated kinship analysis, the effectiveness at the threshold of 4/-4 reached 98.39% for relationship parent-child vs. unrelated individuals, and the effectiveness at the threshold of 2/-2 for relationship full-sibling vs. unrelated individuals was 93.01%, which was greater than that of 15 STR loci (86.75% for relationship parent-child vs. unrelated individuals and 81.73% for relationship full-sibling vs. unrelated individuals). After combining our 29 microhaplotype loci with other 50 short and highly polymorphic microhaplotype loci, the effectiveness values at the threshold of 2/-2 were 82.42% and 90.89% for relationship half-sibling vs. unrelated individuals and full-sibling vs. half-sibling. The short and highly polymorphic microhaplotype panel we developed may be very useful for paternity testing and full sibling testing in degraded samples, and in combination with short and highly polymorphic microhaplotype loci reported by other researchers, may be helpful to analyze more distant kinship relationships.

Broad-Specificity Aptamer of Sulfonamides: Isolation and Its Application in Simultaneous Detection of Multiple Sulfonamides in Fish Sample.

Sulfonamide antibiotics (SAs) are widely used in animal husbandry and aquaculture, and the excess residues of SAs in animal-derived foods will harm the health of consumers. In reality, various SAs were alternately used in animal husbandry and aquaculture, and thus, it is urgent need to develop simple and high-throughput methods for simultaneously detecting multiple SAs or groups of SAs in order to realize rapid screening of total SAs residues in animal-derived foods. We herein isolated a broad-specificity aptamer for SAs by using a multi-SAs systematic evolution of ligands by exponential enrichment (SELEX) strategy. The isolated broad-specificity aptamer has a higher binding affinity to five different SAs including sulfaquinoxaline (SQ), sulfamethoxypyridazine (SMPZ), sulfametoxydiazine (SMD), sulfachloropyridazine (SCP), and sulfapyridine (SPD) and, thus, can be used as a bioreceptor for developing various high-throughput methods for the simultaneous detection or rapid screening of above five SAs. Based on the isolated broad-specificity aptamer and Cy7 (diethylthiatricarbocyanine) displacement strategy, a colorimetric aptasensor was developed for the simultaneous detection of SQ, SMPZ, SMD, SCP, and SPD with a visual detection limit of 2.0-5.0 µM and a spectrometry detection limit of 0.2-0.5 µM. The colorimetric aptasensor was successfully used to detect SQ, SMPZ, SMD, SCP, and SPD in fish muscle with a recovery of 82%-92% and a RSD (n = 5) < 7%. The success of this study provided a promising bioreceptor for developing various high-throughput methods for on-site rapid screening of multiple SAs residues, as well as a simple method for the rapid and cost-effective screening of total SQ, SMPZ, SMD, SCP, and SPD in seafood.