Prime editing for ocular gene therapy and disease modeling: a narrative review of advances, delivery, and translational readiness.

Telomerase reverse transcriptase promoter (TERTp) mutations are associated with aggressive thyroid cancer and are most frequently found in anaplastic and poorly differentiated thyroid cancer. Pre-operative thyroid nodule molecular testing can detect TERTp, denoting a high risk of malignancy and possible aggressive clinical features such as extrathyroidal extension, regional lymph node metastases, and distant metastases. There are two described hot spot point mutations: the more common C228T and a C250T variant. Canonically, these mutations are mutually exclusive and drive monoallelic TERT expression. In this case series, we describe thyroid cancers where both the C228T and C250T variants were detected in preoperative thyroid nodule fine needle aspiration samples sent for Afirma molecular testing. All had co-mutations along with BRAFp.V600E or PIK3CAp.H1047R. Each sample was sent for kinship (relatedness between individuals) analysis to confirm the DNA and RNA samples were from the same patient and not due to sample cross contamination. All cases had confirmed thyroid carcinoma on histopathology after surgical resection. To our knowledge, this is the first report of dual TERTp mutations detected in the preoperative setting in thyroid carcinoma. Clinical correlation with future cases will be of interest, particularly if cases with monoallelic dual TERTp mutations are discovered.

Enhanced rabies surveillance (ERS) of wildlife populations complements passive public health surveillance through active field-based sampling. The combined data augment rabies management decision-making through tracking virus distribution and movement, rapidly identifying potential outbreaks, and informing containment and elimination strategies at regional and landscape scales. Laboratory diagnostic methods are critical to ERS and several gold standard methods are recommended for detection of Lyssavirus rabies (RV); each validated using central nervous system (CNS) tissue which requires necropsy and cold storage, posing challenges and risks in remote field settings. Alternative sample types to CNS tissue may improve ERS efficiency and field personnel safety, provided they offer robust diagnostic specificity and sensitivity. We evaluated multiple sample types opportunistically collected postmortem from naturally infected striped skunks (Mephitis mephitis) and used real-time reverse transcriptase PCR (rtRT-PCR) to evaluate diagnostic sensitivity and specificity of each sample type compared to CNS tissue. The detection rate of RV RNA was 97 % (95 % CI 81-100) for oral swabs and 96 % (95 % CI 80-100) for eye swabs; when swab types were combined, the detection rate was 100 % (95 % CI 85-100) and comparable to detection from CNS. Other sample types demonstrated compromised diagnostic sensitivities (33-76 %). All sample types were 100 % specific for RV diagnosis. The combination of eye and oral swabs as ERS samples may expand sampling opportunities that improve efficiency, mitigate sample collection and storage challenges, and decrease RV exposure risk among field staff while enhancing the information provided to estimate impacts of RV management on target wildlife populations.

Bone is a dynamic tissue that is maintained through the co-ordination of bone resorption and bone formation. An imbalance of these processes can lead to bone disease. In vitro studies of osteoblasts can help to understand bone formation, but primary cells have a limited lifespan in culture. Herein, we report the successful generation of equine immortalized osteoblasts through the stable overexpression of human telomerase reverse transcriptase (hTERT) and Simian virus 40 (SV40) large T-antigen in osteoblasts isolated from trabecular bone taken from the third metacarpal of a two-year-old Thoroughbred horse. Primary osteoblasts displayed limited proliferation in culture, a decrease in the expression of osteogenic-associated genes and alkaline phosphatase activity with increasing passage and a failure to survive and produce a mineralised matrix after 21 days of osteogenic culture at high passage. In contrast, immortalized equine osteoblasts could be expanded for over 50 passages while retaining osteogenic gene expression, high alkaline phosphatase activity, a normal karyotype and the ability to produce a mineralised matrix after osteogenic culture. The immortalized equine osteoblasts therefore constitute a useful in vitro model to study equine bone formation.

The role of retrotransposons at the interface of DNA damage and the innate immune response.

Design, synthesis, ADMET profiling, POM analysis and molecular dynamics studies of coumarin-DAPY derivatives as HIV-1 reverse transcriptase inhibitors

MK-8527 is a novel oral nucleoside reverse transcriptase translocation inhibitor under clinical development as HIV-1 prevention. Two Phase 1 single-dose monotherapy studies were conducted to evaluate antiretroviral activity, pharmacokinetics (PK), and safety of MK-8527 in adults living with HIV-1 who had not previously taken antiretroviral agents. In two Phase 1 studies, participants received a single oral dose of MK-8527 (0.25, 0.5, 1, 3, or 10 mg). Reduction in viral load (measured as log10 plasma HIV-1 RNA copies/mL at 7 days post-dose), PK of plasma MK-8527 through 7 days, intracellular MK-8527-triphosphate (TP, the active form of MK-8527) through 28 days, exposure-response relationship, and safety through 28 days were assessed. Adverse events were descriptively summarized. In total, 37 participants completed the studies. After single doses of MK-8527 0.5 to 10 mg, the mean decrease in HIV-1 RNA at 7 days-post dose was ≥1.0 log10 copies/mL. The inhibitory quotient (defined as the ratio of MK-8527-TP concentration at 168 hours post-dose [C168] to the mean intracellular concentration of MK-8527-TP at the half-maximal inhibitory concentration [IC50]) exceeded 3 at single doses of ≥0.5 mg. MK-8527 at all dose levels was well tolerated, with a limited number of mild or moderate adverse events that were determined by investigators to be unrelated to the study treatment. In adults living with HIV-1 who had not previously taken antiretroviral agents, single doses of MK-8527 as low as 0.5 mg achieved ≥1.0 log10 decreases in HIV-1 RNA at 7 days post-dose administration. www.clinicaltrials.gov NCT03615183, NCT05494736.

Islatravir once monthly (qm), a nucleoside reverse transcriptase translocation inhibitor with a long half-life, was evaluated for safety and tolerability in cisgender men and transgender women who have sex with men and are at increased likelihood of HIV-1 exposure. IMPOWER-24 (NCT04652700) was a double-blind, Phase 3 study. Participants were randomized 2:1 to islatravir 60 mg oral qm or emtricitabine (FTC; 200 mg) coformulated with either tenofovir disoproxil (245 mg) or tenofovir alafenamide (TAF; 25 mg) once daily (qd). After ∼9 months, blinded islatravir was discontinued due to lymphocyte reductions; participants were offered open-label FTC/tenofovir disoproxil or FTC/TAF qd for 20 months. 494 participants were enrolled (328 islatravir; 166 comparator): 91.5% were cisgender men, 41.7% were White, and median age was 27 years. Mean blinded dosing duration was 4.7 months (islatravir) versus 4.3 months (comparator). 211 participants (64.3%) in the islatravir group and 128 (77.1%) in the comparator group had ≥1 adverse event (AE). Most AEs were mild or moderate, with one AE leading to product discontinuation (islatravir; gastroesophageal reflux). Serious AEs occurred in <2%; none were related to study product. Change in total lymphocytes in the islatravir group at Month 3 was -7.4%; a trend toward recovery was observed after islatravir was stopped. Mean total lymphocytes remained within normal range. No HIV-1 infections occurred in either group during the double-blind phase. Islatravir qm was generally well-tolerated; decreases in total lymphocytes were observed with islatravir. Original primary efficacy objectives were not assessed due to early study stoppage.

Human telomerase reverse transcriptase (hTERT) is a key determinant of telomere maintenance and cellular aging. Oxidative stress plays a role in neurodegenerative processes by causing cellular damage through increased reactive oxygen species. Our study aimed to reveal the relationship between hTERT and oxidative stress in the pathophysiology of epilepsy. The study included 45 individuals diagnosed with epilepsy and 55 healthy controls. hTERT concentration, total oxidant status (TOS), total antioxidant status (TAS), superoxide dismutase (SOD), and thiol-disulfide homeostasis were measured in serum samples. A significant decrease in hTERT levels, an increase in oxidative stress markers (TOS, OSI), and a decrease in antioxidant levels (TAS, SOD), and total and native thiol levels were determined in epilepsy patients. In addition, a significant and negative correlation was found between hTERT and native thiol. Our study suggested that the interaction between oxidative stress and hTERT levels in the pathophysiology of epilepsy may play an important role in seizure generation and progressive neural damage. This study will form the basis for further research and guide the identification of potential biomarkers for treating epilepsy. We measured the levels of a protective protein that helps preserve chromosomes and several markers of cell stress in people with epilepsy and healthy volunteers. People with epilepsy showed lower levels of this protective protein, weaker antioxidant defenses, and higher oxidative stress. We also found that lower protein levels were linked to fewer molecules that guard cells from damage. These changes may help explain why seizures occur and why the brain can suffer ongoing injury in epilepsy.

Virological failure (VF) with long-acting injectable cabotegravir and rilpivirine (CAB/RPV-LA) is uncommon but often associated with selection of resistance, potentially limiting future treatment options. Registration trials associated VF risk with baseline RPV resistance, A1/A6 subtypes, and body mass index (BMI) >30 kg/m2, but these factors have rarely been analyzed in other clinical settings. We summarize the first ∼100 reported VF cases, focusing on subtypes, drug levels, and resistance patterns. Published data on CAB/RPV-LA through July 2025 were analyzed for risk factors. Resistance mutations were interpreted using the Stanford HIVdb database. After excluding duplicates, 94 VF cases were analyzed. Only 4.4% met the high-risk threshold of ≥2 risk factors. Subtype A lineages were reported in 26.4%, preexisting RPV mutations in 14.7%, and BMI >30 kg/m2 in 36.9%. At failure, low CAB or RPV levels were observed in 29% but did not differ from treatment successes. Predicted reduced susceptibility to CAB or RPV was observed in 87.2% (56% for both), with CAB resistance mutation N155H more frequently observed among subtype A lineages. Predicted susceptibility to dolutegravir/bictegravir (44.3%), doravirine (39.7%), or etravirine (35.9%) was common, but high-level resistance was rare. Emergent resistance in VF cases often resulted in cross-resistance to other nonnucleoside reverse transcriptase inhibitors and integrase strand transfer inhibitors. Although most cases did not meet the high-risk profile as defined by registration trials, subtype A lineages were overrepresented. Low drug levels were not elevated versus treatment successes. These data suggest that subtype-specific factors beyond A6 may influence VF risk and merit further study.

N6-Methyladenosine (m6A) on mature mRNA has been extensively characterized, yet its precise mapping and functions in nuclear noncoding RNAs remain elusive. To address this issue, we recently developed Nuclear-m6A-label-seq, a metabolic labeling-based method for transcriptome-wide nuclear m6A profiling at single-base resolution. This approach builds on the prototypical m6A-label-seq principle, in which an allyl group, instead of methyl group, is metabolically installed at N6-position at supposed RNA m6A-generating adenosines and the resultant N6-allyl adenosine is subsequently converted into 1, N6-cyclized adenosine (cyc-A) by mild iodination reaction. During RNA reverse transcription, HIV reverse transcriptase is employed to introduce a base misincorporation at cyc-A sites while enabling a template switch to incorporate adapter sequences to the complementary DNA end in a single step. Through this strategy, library construction is shortened to about 6 h, and the required cell-labeling total RNA input is reduced to 5 μg of total nuclear RNA, representing a 100-fold reduction compared to the prototypical protocol. Both polyadenylated and nonpolyadenylated nuclear transcripts are captured through the sequential nuclear RNA isolation and rRNA depletion. Following high-throughput sequencing, reads from human cells are aligned with the complete T2T-CHM13 genome, enabling accurate mapping of repetitive regions. Aligned reads are then analyzed using the user-friendly rMATS-DVR pipeline to identify high-confidence m6A sites based on cyc-A-induced misincorporation patterns. Here, we provide a detailed step-by-step protocol for Nuclear-m6A-label-seq, which stands for a direct and high-resolution approach for profiling the nuclear m6A epitranscriptome.

Cervical carcinoma, exhibits distinctive hallmarks, including sustained proliferation and replicative immortality. Ki-67 is a well-established marker of cell proliferation, while the presence of telomerase, particularly its catalytic subunit human Telomerase Reverse Transcriptase (hTERT), is associated with the uncontrolled proliferation seen in many cancers. The expression of Ki-67 and the hTERT component of telomerase was investigated in various cervical lesions. Tissue microarray blocks were prepared from a total of 586 paraffin-embedded cervical tissue specimens received at Sultan Qaboos University Hospital and Royal Hospital between January 2010 and December 2018. Immunohistochemistry was performed to assess the expression of both markers. The results showed that Ki-67 expression increased significantly with the severity of cervical lesions (p < 0.05), with SCC displaying high Ki-67 expression in more than 50% of tumor cells. However, statistical analysis revealed no significant correlation between Ki-67 expression and lesion prognosis. On the other hand, hTERT showed a significantly higher expression in low-grade LSIL (p < 0.05), whereas high-grade squamous intraepithelial lesions and SCC predominantly showed negative hTERT expression. hTERT staining was mainly localized to the nucleus across all cervical lesions, with some cytoplasmic and combined expressions, and it was generally of mild intensity; however, this correlation was not statistically significant. Additionally, the percentage of hTERT-positive cells was mostly below 10% in all lesion types, with no statistically significant differences observed. Our findings suggest that the use of Ki-67 and hTERT component of telomerase combination might not be sufficient to predict the prognosis of cervical lesions. Nonetheless, the observed expression patterns of each biomarker indicate a potential role in early carcinogenesis.

EXPRESS: Large cell lymphoma in four cats after successful treatment of feline infectious peritonitis with oral GS-441524 - a novel clinical observation.

The developmental dysfunction of cartilage progenitor cells (CPCs) causes dwarfism. Radical therapies for dwarfism remain underdeveloped. Recently, the therapeutic benefits of extracellular vesicles (EVs) released from human exfoliated deciduous tooth-derived stem cells (SHED) have been investigated for their potential to restore disease-target cells. However, the effects of EVs on human CPCs for the treatment of dwarfism remain unclear. We investigated the impact of EVs on cell proliferation, telomerase activity, telomerase reverse transcriptase (TERT) expression, cell cycle, and extracellular signal-regulated protein kinase 1/2 (ERK1/2) levels in human CPCs and in our established osteogenesis imperfecta (OI)-specific SHED (OI-SHED). EVs enhanced the proliferation and G1/S phase transition of CPCs, which was associated with increased TERT expression and telomerase activity. However, RNase-preconditioned EVs did not attenuate the efficacy of EVs in CPCs. ERK1/2 inhibitor tests demonstrated that CPCs exhibited suppressed proliferation, telomerase activity, and G1/S phase progression. EV-transferring CD29 induced ERK1/2 phosphorylation in CPCs, subsequently activating telomerase activity to induce proliferation. Interestingly, EV stimulation restored the reduction in cell proliferation, cell cycle progression, phosphorylated ERK1/2 levels, and TERT expression in OI-SHED. In conclusion, the present findings provide new insights into ERK1/2-mediated proliferation of human CPCs using EVs.

HIV-1 treatment has advanced with various antiretroviral regimens. Although efavirenz (EFV)-based regimens have been widely used, current guidelines recommend integrase strand transfer inhibitor (INSTI)-based therapy as first-line. However, INSTI may cause weight gain and adverse lipid changes, creating new unmet metabolic needs. Novel agents like ainuovirine (ANV), a non-nucleoside reverse transcriptase inhibitor (NNRTI), may offer effective virological suppression (VS) with improved tolerability, but their long-term real-world safety and effectiveness remain unclear. This was a multicenter, retrospective observational cohort study. Virologically suppressed adults on a tenofovir disoproxil fumarate (TDF)/3TC+EFV regimen were either switched to TDF/3TC+ANV (ANV group) based on the physician's discretion or continued on TDF/3TC+EFV (EFV group). Baseline demographic and clinical data were collected, and participants were followed for 48 weeks. The primary effectiveness outcome was the proportion of patients achieving HIV-1 RNA levels below the limit of quantification (LOQ) at week 48. Secondary outcomes included absolute or percentage changes from baseline in CD4+ T-cell count, CD4+/CD8+ ratio. Key secondary safety outcomes included absolute changes from baseline in body weight, BMI, fasting lipid profiles (total cholesterol [TC], triglycerides [TG], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C]), and parameters of liver and renal function. A total of 350 participants who completed the 48-week follow-up were included, comprising 170 patients in the ANV group and 180 patients who remained on EFV. At week 48, the proportion of VS was 96.5% in the ANV group and 96.1% in the EFV group (difference: 1.00 percentage points; 95% CI: -2.77 to 2.77), confirming non-inferiority. No significant differences in CD4+ T-cell recovery or CD4+/CD8+ ratios were observed. The ANV group experienced significantly less weight gain than the EFV group (estimated treatment difference [ETD]: -0.79 kg; P < 0.001). A corresponding trend in BMI change was observed but did not reach statistical significance. ANV also led to more favorable lipid changes, including a significant reduction in total cholesterol (ETD: -0.52 mmol/L; P < 0.001) and triglycerides (ETD: -0.83 mmol/L; P < 0.001) compared to EFV. Liver and renal function profiles remained stable in both groups. Switching from EFV- to an ANV-based regimen effectively maintained VS and led to improved metabolic parameters, including less weight gain and a more favorable lipid profile. As an alternative switch strategy, the ANV-based regimen may be a more beneficial option for people living with HIV (PLWH) who are at high risk of weight-related or dyslipidemia-associated comorbidities.

The BioFire® Global Fever Panel (GFP; BioFire Diagnostics, Salt Lake City, UT) is an assay designed for detecting multiple pathogens, including dengue virus (DENV; Orthoflavivirus denguei). Although its performance has been primarily assessed using composite reference standards (CRS) or through direct comparisons with other tests, its real-world accuracy remains uncertain. The performance of the GFP in combination with real-time reverse transcriptase polymerase chain reaction (qRT-PCR) testing is evaluated in the present study using dengue surveillance data from 224 individuals with acute fever (≤5 days) in a dengue-endemic area of the Peruvian Amazon. Dengue virus RNA was detected in serum and whole blood using qRT-PCR testing and the GFP, respectively. Two reference standards were developed: one based on CRS and another based on latent class analysis. The best latent class model (LCM) included a viral load surrogate. Using qRT-PCR testing or the LCM as a reference, the GFP exhibited a sensitivity of 94.5% (95% CI: 88.4-98.0%) and a specificity of 75.7% (95% CI: 66.8-83.2%). With CRS, the GFP displayed a sensitivity of 95.6% (95% CI: 90.7-98.4%) and 100% specificity. The simultaneous and sequential two-test algorithms for negative yielded comparable performance and exhibited high sensitivity compared with other two-test sequential algorithms. The sequential testing algorithm, which used qRT-PCR testing followed by the GFP for negative results, offered the best balance in cost, time, and performance (sensitivity: 99.3-100%; specificity: 75.7-100%). Although the GFP is more expensive than qRT-PCR testing, it offers a shorter turnaround time per sample. Overall, the findings suggest that the GFP is a reliable and high-performing tool for DENV testing, but its cost may restrict its use in resource-limited settings.

Objective. To perform molecular genetic monitoring of HIV-1 drug resistance to integrase inhibitor (INIs) and to assess the prevalence of mutations associated with viral resistance to these drugs in the Volga Federal District (VFD) from 2021-2024. Materials and methods. The study analyzed 121 blood plasma samples from HIV-infected patients experiencing virological failure of antiretroviral therapy (ART) with integrase inhibitors. The samples were delivered for HIV-1 drug resistance testing from ten subjects of the Volga Federal District: the Republics of Bashkortostan, Mari El, Mordovia, Udmurtia and Chuvashia, and Kirov, Nizhny Novgorod, Penza, Saratov and Ulyanovsk oblasts. HIV-1 genotyping was performed by fragment sequencing of reverse transcriptase, protease, and integrase regions of the HIV-1 pol gene. Information about the samples was obtained from test requisition forms for HIV-1 drug resistance testing provided by the regional AIDS prevention and control centers of the Volga Federal District. Results. The number of HIV-infected patients receiving INIs-based antiretroviral therapies in VFD is increasing, reaching 22.1 % of all treated patients in 2023. The analysis of HIV-1 resistance to INIs revealed mutation combinations associated with resistance to second–generation INIs: R263RK, E138K+T66A+G118R, E138K+G140A+Q148R, E138K+G140A+S147G, S147G+N155H. The following risk factors for developing INIs resistance were determined: the HIV infection duration more than 15 years from the first positive Western blot result (p = 0.057), the age of HIV-infected patients over 35 years (p = 0.079), and existence of HIV-1 resistance to nucleoside reverse transcriptase (NRTIs), non-nucleoside reverse transcriptase (NNRTIs), or protease inhibitors (PIs) (p 0.0001). Conclusions. The growing proportion of HIV-infected patients receiving INIs-based antiretroviral therapies, including second–generation INIs, in the VFD underscores the necessity for regular molecular and genetic monitoring of HIV-1 resistance to INIs which is essential for further study of the prevalence of resistant viral strains to increase the effectiveness of ART.

To examine changes in weight after initiating different dolutegravir-based second-line antiretroviral regimens and to measure the impact of observed weight change on blood pressure and serum lipids. Secondary analysis of the D2EFT trial, a randomised open-label trial in people with HIV. In D2EFT, ritonavir-boosted darunavir plus dolutegravir (DTG + DRV/r) and dolutegravir with tenofovir disoproxil fumarate plus either lamivudine or emtricitabine (DTG + TDF/XTC) were compared to ritonavir-boosted darunavir plus two nucleoside reverse transcriptase inhibitors (DRV/r + 2NRTIs). We used linear mixed effects models for weight, blood pressure and lipid changes and generalized estimating equations for ≥5% weight gain. 826 participants were included. Significantly greater body weight and BMI gains at 48 and 96 weeks were observed in individuals taking DTG + DRV/r or DTG + TDF/XTC than in individuals taking DRV/r + 2NRTIs. Treatment arm (DTG + DRV/r and DTG + TDF/XTC), female gender, Black and Hispanic/Latino ethnicity, lower baseline CD4 count, and higher baseline HIV RNA were all associated with weight gain in multivariable analysis. There was no significant difference between treatment arms in blood pressure changes at 96 weeks after adjustment for weight gain. Both darunavir containing arms demonstrated greater increases in LDL cholesterol than DTG + TDF/XTC, with the greatest increase in the non-NRTI containing arm. Dolutegravir-based second-line regimens were associated with weight gain, which was further influenced by gender, ethnicity and HIV-related factors. DTG-based second-line regimens had no significant effect on blood pressure at 96 weeks; DTG + TDF/XTC was associated with a more favourable lipid profile than darunavir-containing regimens. NCT03017872.

Synthesis and functional characterization of a recombinant HIV-1 reverse transcriptase variant carrying twelve drug-resistance mutations as a platform for antiretroviral screening.

The aim : to perform surveillance over HIV-1 drug resistance mutations among people living with HIV without history of antiretroviral therapy as well as among those receiving antiretroviral treatment and residing in different territories of the Far Eastern Federal district. Materials and methods . A total number of 420 patients diagnosed with HIV-infection and residing in 8 constituent entities of the Far Eastern Federal district were examined. «AmpliSense® HIV-Resist-Seq» kit was used to perform sequencing of the amplified fragments of HIV-1 pol-gene coding protease and a part of reverse transcriptase to detect drug resistance mutations. Stanford University HIVdb Program was employed to retrieve information on drug-resistance mutations. Results and discussion : sub-subtype A6, which is prevalent in Russia, was also dominant in the surveyed group of patients and was isolated in 282 cases (67.1%). A total number of 49 samples were typed as subtype B (11.7%), 12 samples as subtype C (2.9%), 4 samples as subtype G (1.0%). Subtype A7 (0.2%) was detected in one patient from the Republic of Sakha (Yakutia). Different recombinant forms of the virus were identified in 72 patients (17.1%). Percentage of recombinant forms derived from subtypes A and G totaled 80.6% (n=58). Surveillance drug resistance mutations were revealed in 108 out of 248 patients undergoing antiretroviral treatment (43.5%) and in 6 out of 172 treatment-naïve patients (3.5%). Drug resistance to non-nucleoside inhibitors of reverse transcriptase drugs was most common in both treatment naïve patients and those with a prior experience of antiretroviral therapy. Conclusion : regular surveillance of acquired drug resistance provides insight into the effectiveness of HIV prevention and treatment programs in the constituent entities of the Russian Federation and will allow the development of recommendations for treatment strategies.