A developmental validation of the Quick TargSeq 1.0 integrated system for automated DNA genotyping in forensic science for reference samples.

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8 h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2 h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10 ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

Population pharmacokinetic modeling and simulation for nirmatrelvir exposure assessment in Chinese older patients with COVID-19 infection.

Nirmatrelvir is an effective component of Paxlovid, the first oral antiviral drug granted emergency use authorization by the FDA. Nirmatrelvir is prescribed extensively in older adult patients to treat the coronavirus disease 2019 (COVID-19) infection. In this study, population pharmacokinetic modeling with clinical study data was employed to explore the pharmacokinetic profile of nirmatrelvir in older adult Chinese patients with COVID-19 infection. The result suggests that the pharmacokinetic profile of nirmatrelvir can be described by a one-compartment model with first-order absorption and elimination in this study population. The calculated apparent clearance (CL/F), apparent volumes of distribution (V/F), and absorption rate constant (ka) for the typical patient were 4.16 L/h, 39.1 L, and 0.776, respectively. The area under the curve (AUC) of nirmatrelvir in the typical Chinese older adult was approximately three-fold higher than the AUCs in Chinese and Western young adult volunteers. At the same doses, the simulated AUCs were increased by 26%, 43%, 72%, and 135% in virtual populations with creatinine clearances of 60, 45, 30, and 15 mL/min, respectively. Our research provides an instructive reference for nirmatrelvir dose selection in older Chinese adults.

A cowpea severe mosaic virus-based vector simplifies virus-induced gene silencing and foreign protein expression in soybean.

BACKGROUND: Soybean gene functions cannot be easily interrogated through transgenic disruption (knock-out) of genes-of-interest, or transgenic overexpression of proteins-of-interest, because soybean transformation is time-consuming and technically challenging. An attractive alternative is to administer transient gene silencing or overexpression with a plant virus-based vector. However, existing virus-induced gene silencing (VIGS) and/or overexpression vectors suitable for soybean have various drawbacks that hinder their widespread adoption. RESULTS: We describe the development of a new vector based on cowpea severe mosaic virus (CPSMV), a plus-strand RNA virus with its genome divided into two RNA segments, RNA1 and RNA2. This vector, designated FZ, incorporates a cloning site in the RNA2 cDNA, permitting insertion of nonviral sequences. When paired with an optimized RNA1 construct, FZ readily infects both Nicotiana benthamiana and soybean. As a result, FZ constructs destined for soybean can be first delivered to N. benthamiana in order to propagate the modified viruses to high titers. FZ-based silencing constructs induced robust silencing of phytoene desaturase genes in N. benthamiana, multiple soybean accessions, and cowpea. Meanwhile, FZ supported systemic expression of fluorescent proteins mNeonGreen and mCherry in N. benthamiana and soybean. Finally, FZ-mediated expression of the Arabidopsis transcription factor MYB75 caused N. benthamiana to bear brown leaves and purple, twisted flowers, indicating that MYB75 retained the function of activating anthocyanin synthesis pathways in a different plant. CONCLUSIONS: The new CPSMV-derived FZ vector provides a convenient and versatile soybean functional genomics tool that is expected to accelerate the characterization of soybean genes controlling crucial productivity traits.

Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5'-to-3' direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5' terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5' CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5'-to-3' degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.

Synthesis and Characterization of a Series of Temozolomide Esters and Its Anti-glioma Study.

Temozolomide is a first-line therapeutic drug for glioblastoma (GBM), and it has a low solubility, short biological half-life, and resistance to drug limits in clinical applications. Therefore, it is necessary to find more effective anti-tumor drugs to overcome drug resistance and enhance its anti-glioma activity. We therefore used n-butanol, n-hexanol, n-octanol, 1-dodecanol and 1-hexadecanol to synthesize a series of temozolomide ester compounds (TMZEs) and then investigated their physicochemical properties and anti-glioma efficacy. Our results showed that TMZEs had a higher lipophilicity compared to TMZ and could stably exist in plasma and brain homogenates. TMZEs had significantly increased cytotoxicity and cellular uptake in C6 glioma cells as chain lengths increased. Additionally, the IC50 of TMZ-16E towards TMZ-resistant cells (T98G) was 85.9-fold lower than that of TMZ (p < 0.001), and Western blot results demonstrated that TMZ-16E could significantly reduce the expression of O6-methylguanine-DNA-methyltransferase (MGMT). The in vivo anti-glioma efficacy of TMZ-16E were then investigated in orthotopic and subcutaneous GBM models. TMZ-16E prolonged the survival time to 35 days in orthotopic glioma bearing rats, which was 1.94-fold longer than the survival time of rats treated with TMZ, and TMZ-16E increased tumor cell apoptosis based on TUNEL staining. Moreover, TMZ-16E (50 mg/kg) noticeably slowed the growth of T98G subcutaneous tumors by down-modulating MGMT expression in subcutaneous GBM-bearing mice, indicating that TMZ-16E could effectively reverse drug resistance. In conclusion, TMZEs improved the lipophilicity and stability of these drugs. Especially, TMZ-16E could reverse drug resistance and improve therapeutic effects of TMZ, which has clinical application potential for GBM treatment.

[Regulation of acupuncture on expression of AKT protein in lung tissues of asthma rats].

OBJECTIVE: To explore the effects of acupuncture on the expression of protein kinase B (PKB/AKT) in lung tissues of asthma rats. METHODS: Forty SPF male SD rats were randomly divided into a blank group, a model group, an acupuncture group and a blocker group, 10 rats in each one. The rat model of asthma was established by egg albumin stimulation in the model group, acupuncture group and blocker group. Since the establishment of rat model, the rats in the acupuncture group were treated with acupuncture at "Dazhui" (GV 14), "Feishu" (BL 13) and "Fengmen" (BL 12) before atomization; the rats in the blocker group were treated with intervention of blocker LY294002, once every two days, for 7 times. There was no treatment in the blank group and model group. HE staining was applied to observe the morphologic changes of lung tissues; the immunohistochemical method was applied to test the protein expression of AKT in lung tissue. RESULTS: HE staining indicated the infiltration and aggregation of a variety of inflammatory cells around airways, as well as bronchial smooth muscle spasm and confined lumen in the model group; in the acupuncture group and blocker group the inflammatory cells were less and confined lumen was relieved. Compared with the blank group, the protein expression of AKT was higher in the model group (P<0.05); compared with the model group, the protein expression of AKT in the acupuncture group and blocker group was reduced (both P<0.05); the differences between the acupuncture group and blank group, blocker group were not significant (both P>0.05). CONCLUSIONS: Acupuncture could reduce the protein expression of AKT in lung tissue in asthma rats, leading to relieved inflammation reaction and airway remodeling.

[Effect of Acupuncture on c-fos Expression in the Lung Tissue of Bronchial Asthmatic Rats].

Objective To observe the effect of acupuncture on c-fos expression in the lung tissue of asthmatic rats. Methods Totally 70 SPF grade male SD rats were randomly divided into 7 groups, i.e., the blank group (A) , the asthma model group (B) , the blank control group (C) , the asthma-model acupuncture control group (D) , the asthma model acupuncture group ( E) , the asthma model sham-acu- puncture group (F) , the blank acupuncture group (G) , 10 rats in each group. Corresponding interventions were performed to each group. The protein expression of c-fos in lung tissue of rats was detected u- sing immunohistochemistry and Western blot respectively. Results Immunohistochemistry showed negative expression of c-fos protein in Group A, C, G, and E, and weakly positive in Group B, D, and F. Results of Western blot showed the protein expression of c-fos was higher in Group B than in Group A and E (P <0. 01). The protein expression of c-fos was lower in Group E than in Group D and F (P <0. 05, P < 0. 01). Conclusion Acupuncture could reduce the protein expression of c-fos in lung tissue, thus attenu- ating inflammation reaction.

Dexmedetomidine prevents post-ischemic LTP via presynaptic and postsynaptic mechanisms.

Increasing evidence indicates that dexmedetomidine (DEX), a selective α2-adrenergic receptor agonist, has a neuroprotective effect against cerebral injury. However, it remains unknown whether and how DEX functionally prevents the pathological form of synaptic plasticity caused by ischemia in the hippocampal CA1 neurons. To address this issue, we analyzed the role of DEX using a model of brain ischemia (oxygen and glucose deprivation, OGD) referred to as post-ischemic LTP (i-LTP). We found that DEX could reduce i-LTP by selectively activating α2 receptors. To clarify its detailed mechanisms, the presynaptic and postsynaptic roles of DEX were investigated. The activation of the α2 receptors of DEX decreased the frequency spontaneous mEPSCs, which exerted its presynaptic mechanisms. In addition, DEX also decreased the amplitude of mEPSCs and prevented the depolarization of postsynaptic membranes during OGD treatment, which exerted its postsynaptic mechanisms. More importantly, our results indicate that postsynaptic ß receptors, not α1 receptors, participated in i-LTP. Therefore, these results demonstrated that decreasing ß receptors activation by DEX-medicated pre- and post-synaptic α2 receptors activation is responsible for i-LTP. Because of the NMDARs required for i-LTP, we further examined the critical roles of postsynaptic ß receptors downstream PKA regulation of NMDA receptor-mediated EPSCs (NMDA EPSC). We clarified that it is attributable to the direct effect of DEX on NMDA EPSC as mediated by PKA inactivation. These findings suggest that DEX can protect neurons from functional damage caused by a relatively mild degree of transient cerebral ischemia, and this effect is mediated by both presynaptic reduction of NE and glutamate release and postsynaptic suppression of NMDAR activation by ß receptors and downstream PKA regulation.

A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 µL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 µL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 µm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

[Low volume amplification in single cell separation and inspection].

OBJECTIVE: To establish optimal amplification conditions with the application of 0.2 mL test tube in single cell separation and inspection. METHODS: Oral epithelium cell suspension was prepared. Five or ten cells were collected with 0.2 mL test tube. Then DNA were amplified with Identifiler Plus kit in three different conditions in which the proteinase K addition, gold enzyme concentration in PCR reaction, and PCR reaction cycles were adjusted separately. Finally the detection rate, allelic dropout rate and artificial alleles were compared among the groups. RESULTS: In these 3 different conditions: addition of proteinase K, addition of 0.4 microL gold enzyme in PCR reaction, and use of 32 cycles, the detection rate was higher and allelic dropout rate was lower than the other conditions. CONCLUSION: In single cell separation and inspection, the usage of 0.2 mL test tube could be an effective supplement to chip-low volume amplification.