Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5\'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer\'s chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

BACKGROUND: In the context of primary HPV cervical cancer screening, the identification of minor screening abnormalities necessitates triage tests to optimize management and mitigate overtreatment. Currently, reflex cytology and reflex p16/Ki67 dual-stain (DS) are under scrutiny for their applicability in primary HPV-based screening. However, there remains a dearth of comprehensive data for comparing their performance.
METHODS: Among 30,066 results from liquid-based cervical cancer screening tests, a cohort of 332 cases was meticulously selected based on available high-risk human papillomavirus (HPV) test results, limited genotyping for HPV 16 and 18, liquid-based cytology, DS, and histology outcomes from standardized colposcopy with biopsy. For cases positive for 12 other high-risk HPV genotypes, three retrospective triage approaches were analyzed. We computed the positive predictive value (PPV) for the detection of high-grade squamous intraepithelial lesions or worse (HSIL+).
RESULTS: Both triage models employing DS (reflex cytology followed by DS and reflex DS alone in all cases) exhibited significantly higher PPV for HSIL+ compared to the strategy with reflex cytology alone (35.9%/33.3% vs. 18.8%; p < 0.0001). Additionally, these DS-based models showed higher negative predictive values (NPV) (100%/96.2% vs. 69.2%; p = 0.0024/0.0079). In the DS-inclusive models, fewer colposcopies were necessitated (103/102 vs. 154), and fewer cases of HSIL+ were overlooked (0/3 vs. 8).
CONCLUSIONS: Our findings suggest that p16/Ki67 dual-stain, either as a standalone or combined triage test, holds promise for the effective detection of HSIL+ in patients with minor screening abnormalities in primary HPV-based cervical cancer screening.

Although the trigger for the neurodegenerative disease process is unknown, the relevance of aging stands out as a major risk for the development of neurodegeneration. In this review, we highlighted the relationship between the different cellular mechanisms that occur as a consequence of aging and transcription factor nuclear factor erythroid-2-related factor 2 (NRF2) and the connection with the TAU protein. We focused on the relevance of NRF2 in the main processes involved in neurodegeneration and associated with aging, such as genomic instability, protein degradation systems (proteasomes/autophagy), cellular senescence, and stem cell exhaustion, as well as inflammation. We also analyzed the effect of aging on TAU protein levels and its aggregation and spread process. Finally, we investigated the interconnection between NRF2 and TAU and the relevance of alterations in the NRF2 signaling pathway in both primary and secondary tauopathies. All these points highlight NRF2 as a possible therapeutic target for tauopathies.

In acute promyelocytic leukemia (APL), increased cell burden in the peripheral blood due to either the disease itself or early treatment with all-trans retinoic acid could cause hyperleukocytosis (HL) before induction chemotherapy. However, therapeutic leukapheresis has seldom been used because of concerns of subsequent coagulopathy after this invasive procedure. The aim of this study was to evaluate the effects of leukapheresis in APL, especially for efficacy and safety.
We retrospectively analyzed newly diagnosed patients with APL from January 2009 to March 2022. Among 323 patients, 85 had white blood cell count above 40 × 109/L before induction chemotherapy. Thirty-nine patients were initially treated with leukapheresis, whereas the other 46 were not. Clinical and laboratory parameters between these groups were compared.
There was a trend toward favorable 30-day survival rate for the leukapheresis group compared with the non-leukapheresis group (76.9% and 67.4%; P = 0.24). The complications including subsequent intensive unit care (P = 0.23), severe hemorrhagic events (P = 0.13) showed no significant differences between the two groups. The patients were divided into subcohorts, and the survival rates of the leukapheresis and non-leukapheresis groups were 92.3% (95% confidence interval [CI], 77.8%-100.0%) versus 58.3% (95% CI, 38.6%-78.1%) (P = 0.03) in \"sequential HL\" and 76.7% (95% CI, 61.5%-91.8%) versus 54.8% (95% CI, 37.3%-72.4%) (P = 0.03) in \"symptomatic HL,\" respectively. Moreover, in the \"sequential HL\" subcohort, the cumulative incidence of differentiation syndrome and following adverse events were significantly lower in the leukapheresis group.
In APL with \"sequential HL\" or \"symptomatic HL\" from either the disease itself or the effect of all-trans retinoic acid, therapeutic leukapheresis could be applied to reduce leukemic cell burden without significant risks.

Healthy pregnancy is characterized by a reduction in total peripheral vascular resistance which produces a decrease in maternal blood pressure. Failure of this normal maternal adaptation to pregnancy results in hypertensive disorders of pregnancy, which are dangerous for both the mother and fetus. Recently, it has been proposed that Dahl salt-sensitive (SS) rats are a model of spontaneous superimposed preeclampsia when maintained on a normal salt diet. Since these reports are from animals that are derived from sources that are not commercially available, the purpose of this study was to evaluate the blood pressure and pregnancy outcomes of SS rats from a commercial vendor. Mean arterial blood pressure was measured by indwelling femoral catheter in anesthetized SS virgin and SS day 21 late pregnant rats from Charles River Laboratories (CRL). Fetal outcomes from SS rats and control Sprague-Dawley (SD) rats were also measured. All rats were euthanized by exsanguination under anesthesia and tissues weighed. Virgin SS rats were found to have normal mean arterial blood pressure (102 ± 2 mmHg), and late pregnant SS rats had the normal decrease in maternal blood pressure. SS rats were also found to have normal pregnancy outcomes. These results suggest that SS rats from CRL are not a model of superimposed preeclampsia. Further studies will be aimed at determining the differences between the blood pressure phenotype and pregnancy outcomes of existing colonies of SS rats in order to elucidate the mechanisms permitting the development of preeclampsia in certain colonies of this strain.

Cannabidiol (CBD) is approved for treatment of Dravet syndrome (DS), Lennox-Gastaut syndrome (LGS), and tuberous sclerosis complex (TSC). Several studies suggest antiseizure effects also beyond these three epilepsy syndromes.
In a retrospective multicenter study, we analyzed the efficacy and tolerability of CBD in patients with epilepsy at 16 epilepsy centers.
The study cohort comprised 311 patients with epilepsy with a median age of 11.3 (0-72) years (235 children and adolescents, 76 adults). Therapy with CBD was off-label in 91.3% of cases due to age, epilepsy subtype, lack of adjunct therapy with clobazam, and/or higher dose applied. CBD titration regimens were slower than recommended, with good tolerability of higher doses particularly in children. Of all patients, 36.9% experienced a reduction in seizure frequency of >50%, independent of their epilepsy subtype or clobazam co-medication. The median observation period was 15.8 months. About one third of all patients discontinued therapy within the observation period due to adverse effects or lack of efficacy. Adverse effects were reported frequently (46.9%).
Our study highlights that CBD has an antiseizure effect comparable to other antiseizure medications with a positive safety profile independent of the epilepsy subtype. Comedication with clobazam was not associated with a better outcome. Higher doses to achieve seizure frequency reduction were safe, particularly in children. These findings call for further trials for an extended approval of CBD for other epilepsy subtypes and for children <2 years of age.

Metabolic and bariatric surgery (MBS) could improve health-related quality of life (HrQoL) for selected patients with obesity. Although biliopancreatic diversion with duodenal switch (BPD-DS) is regarded as the most effective MBS technique in achieving weight loss, no consensus has been reached on the impact of BPD-DS on HrQoL. The aim of this meta-analysis is to assess the mid-term HrQoL after BPD-DS in the management of patients with obesity.
Cochrane, Embase, APA PsycInfo, PubMed, Scopus, and Web of Science were searched for articles from their inception to August 2022 by two independent reviewers using the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) system. The review was registered prospectively with PROSPERO (CRD42022352073).
From 223 studies screened, twelve studies met the eligibility criteria, with a total of 937 patients with obesity undergoing BPD-DS. Minimal clinically important differences (MCID) were reached for the physical component summary score (PCS) of the 36-Item Short-Form Health Survey (SF-36) (MD = 13.4) and impact of weight on quality of life (IWQOL)-Lite total score (MD = 48.7). Similarly, MCIDs were attained in the Laval questionnaire and SF-36 subscales.
Our meta-analysis demonstrated an improvement in mid-term HrQoL after BPD-DS. Despite the promising trends demonstrated in this meta-analysis, further studies with large sample sizes are needed to evaluate the impact of HrQoL on patients with obesity after BPD-DS.

A new series of triclosan (TCL)-mimicking diaryl ether derivatives 7-25 were synthesized and evaluated as inhibitors of enoyl acyl carrier protein reductase InhA enzyme. In addition, these derivatives were screened as inhibitors of drug-susceptible (DS), multidrug-resistant (MDR), and extensive drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains. Most compounds exihibted superior anti-TB activities and improved ClogP compared to TCL as a standard drug. The present work has led to the identification of compounds 14, 19 and 24 which possess remarkable activities against DS, MDR and XDR MTB strains with MIC values of 1.95, 3.9 and 15.63 µg/ml, respectively for compound 14, 1.95, 3.9 and 7.81 µg/ml, respectively for compound 19 and 0.98, 1.95 and 3.9 µg/ml, respectively for compound 24. Most compounds did not exhibit toxicity to HePG2 normal cell line. Compounds 14, 19 and 24, presenting the best MIC values, were further evaluated as inhibitors of InhA enzyme. They showed high binding affinities in the micromolar range with IC50 values of 1.33, 0.6, and 0.29 µM for compounds 14, 19, and 24, respectively. Furthermore, molecular docking approach was utilized to understand the difference in bioactivities between the new compounds. In particular, the results revealed strong binding interactions and high docking scores of compounds 14, 19 and 24, which could correlate with their high activities. Mainly, the molecular modelling study of compound 24 provides an excellent platform for understanding the molecular mechanism regarding InhA inhibition. Thus, compound 24 could be a lead compound for future development of new antitubercular drugs.

Preemptive targeted pharmacogenetic testing of candidate variations in DPYD is currently being used to limit toxicity associated with fluoropyrimidines. The use of innovative next generation sequencing (NGS) approaches could unveil additional rare (minor allele frequency <1%) genetic risk variants. However, their predictive value and management in clinical practice are still controversial, at least partly due to the challenges associated with functional analyses of rare variants. The aim of this study was to define the predictive power of rare DPYD variants burden on the risk of severe fluoropyrimidine-related toxicity. The DPYD coding sequence and untranslated regions were analyzed by NGS in 120 patients developing grade 3-5 (NCI-CTC vs3.0) fluoropyrimidine-related toxicity and 104 matched controls (no-toxicity). The functional impact of rare variants was assessed using two different in silico predictive tools (i.e., Predict2SNP and ADME Prediction Framework) and structural modeling. Plasma concentrations of uracil (U) and dihydrouracil (UH2) were quantified in carriers of the novel variants. Here, we demonstrate that the burden of rare variants was significantly higher in patients with toxicity compared to controls (p = 0.007, Mann-Whitney test). Carriers of at least one rare missense DPYD variant had a 16-fold increased risk in the first cycle and an 11-fold increased risk during the entire course of chemotherapy of developing a severe adverse event compared to controls (p = 0.013 and p = 0.0250, respectively by multinomial regression model). Quantification of plasmatic U/UH2 metabolites and in silico visualization of the encoded protein were consistent with the predicted functional effect for the novel variations. Analysis and consideration of rare variants by DPYD-sequencing could improve prevention of severe toxicity of fluoropyrimidines and improve patients\' quality of life.

BACKGROUND: Discriminative stimuli (DS) are cues that predict reward availability. DS are resistant to extinction and motivate drug seeking even after long periods of abstinence. Previous studies have demonstrated that sign-tracking (ST) and goal-tracking (GT) differences in Pavlovian approach predict distinct cue-modulated vulnerabilities to cocaine reinstatement. GT rats show heightened reinstatement to contextual and DS, while ST rats show heightened reinstatement to discrete stimuli. Here we examine whether DS modulate reinstatement after electric barrier-induced abstinence and whether tracking-related relapse vulnerabilities generalize to opioid relapse.
OBJECTIVE: We examine whether DS-modulated reinstatement to fentanyl seeking persists in the presence of reduced adverse consequences after electric barrier-induced abstinence. We also examine whether tracking differences predict the magnitude of DS-modulated reinstatement of fentanyl seeking after electric barrier-induced abstinence.
METHODS: We used Pavlovian lever autoshaping (PLA) training to determine sign-, goal-, and intermediate tracking groups in male and female Sprague Dawley rats. We then trained rats in a DS model of intermittent fentanyl self-administration, and extinguished drug seeking by imposing an electric barrier of increasing intensity. We then measured the level of DS-modulated reinstatement in the presence of a reduced electric barrier intensity.
RESULTS: We report that DS strongly modulate fentanyl seeking after electric barrier-induced abstinence. DS-modulation of fentanyl acquisition, electric barrier-induced abstinence, and reinstatement was similar for sign- and goal-tracking groups.
CONCLUSIONS: Discriminative stimuli powerfully motivate opioid seeking, despite continued aversive consequences. Pavlovian approach differences do not predict the level of DS-modulated reinstatement to fentanyl seeking after conflict-induced abstinence.