Pharmaceuticals

PurposeExtracellular vesicles (EVs) are increasingly explored as natural vehicles for drug delivery and gene therapy approaches. However, reproducible yield and scalability of EV production still pose major challenges in the clinical translation of EV-based therapies. In this study, we sought to quantify and characterize EVs released by suspension-cultured HEK293 cells (Expi293F cells) grown in shaker flasks or small-scale bioreactors, to investigate how the culturing environment affects EV production yield. MethodsExpi293F cells were cultivated (N=3) in either shaker flasks or a bioreactor system, and total cell density, viability, and size were monitored. Supernatants were drawn daily post-cell seeding and were analyzed for EV quantity, size, morphology, and CD63 expression. ResultsNo significant differences were observed in terms of total cell density, viability, and cell size between both cultivation settings. However, cultivation of Expi293F cells in the bioreactor environment significantly increased EV yield by 3-fold compared to shaker flask cultivation (p < 0.01). Other parameters such as average nanoparticle size, EV morphology, and CD63 expression remained comparable between both cultivation methods. ConclusionThese results demonstrate that Expi293F-derived EV yield can be increased by culturing cells in a scalable bioreactor system. These findings pave the way towards the production of therapeutic-based EVs in a scalable and reproducible manner suitable for future (pre-)clinical applications.

Purpose This study aimed to examine the association between screen time and depressive symptoms among Chinese college students, and to investigate the mediating roles of sleep quality and emotion regulation in this relationship. Furthermore, a serial mediation model was constructed to elucidate the underlying psychological mechanisms linking screen exposure to depression. Methods A stratified cluster sampling method was employed to recruit 10,999 college students for a cross-sectional questionnaire survey. Data were collected on screen time, sleep quality, emotion regulation ability, and depressive symptoms. Descriptive statistics, correlation analyses, and regression analyses were conducted using SPSS 26.0 A serial mediation model was tested using the PROCESS macro (Model 6), and bootstrapping procedures were applied to estimate the significance of indirect effects. Results Correlation analyses indicated that screen time was significantly positively associated with depressive symptoms (r = 0.16, p < 0.01) and sleep quality (r = 0.15, p < 0.01), and significantly negatively associated with emotion regulation (r = -0.13, p < 0.01). Sleep quality was positively correlated with depressive symptoms (r = 0.31, p < 0.01), whereas emotion regulation was negatively correlated with depressive symptoms (r = -0.42, p < 0.01). Regression analyses further showed that screen time significantly positively predicted depressive symptoms ({beta} = 0.712, p < 0.001), positively predicted sleep quality ({beta} = 0.217, p < 0.001), and negatively predicted emotion regulation ({beta} = -0.085, p < 0.001). In addition, both sleep quality ({beta} = 1.318, p < 0.001) and emotion regulation ({beta} = -0.424, p < 0.001) were significant predictors of depressive symptoms. Mediation analyses demonstrated that sleep quality significantly mediated the association between screen time and depressive symptoms (95% CI [0.239, 0.332]), as did emotion regulation (95% CI [0.269, 0.416]). Moreover, a significant serial mediation effect of sleep quality and emotion regulation was observed in the relationship between screen time and depressive symptoms (95% CI [0.082, 0.117]). Conclusion Screen time is significantly associated with depressive symptoms among college students, with sleep quality and emotion regulation serving as important mediating mechanisms. Extended screen exposure may be linked to higher levels of depressive symptoms by impairing sleep quality and weakening emotion regulation capacity.

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5{beta}-dihydrotestosterone (5{beta}-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5{beta}-DHT and related 5{beta}-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5{beta}-DHT and 3{beta}-etiocholanediol (3{beta}-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2, with weaker activity than testosterone, whereas other 5{beta} metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5{beta}-DHT and 3{beta}-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5{beta}-DHT and 3{beta}-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5{beta}-DHT, but not 3{beta}-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5{beta}-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects. HighlightsO_LI5{beta}-DHT and 3{beta}-ecdiol promote AR-dependent prostate cancer cell growth C_LIO_LIBoth are weaker AR agonists than testosterone by RNA-seq and qPCR C_LIO_LISupraphysiologic 5{beta}-DHT suppresses growth via AR-mediated senescence C_LIO_LIGrowth suppression extends to AR mutants W742C and H875Y C_LIO_LI5{beta}-DHT may be a lower-androgenicity testosterone surrogate for BAT C_LI

Background: Diabetes mellitus is an increasing public health challenge in Sierra Leone, where access to diagnosis, treatment, and long-term care remains limited. Traditional medicine continues to play a significant role in disease management; however, ethnobotanical knowledge related to diabetes remains insufficiently documented. Methods: A cross-sectional ethnobotanical survey was conducted among 40 informants, including traditional healers, herbalists, and knowledgeable community members in Waterloo, Pendembu, and Bo. Data were collected using structured questionnaires administered via Kobo Toolbox and paper-based tools. Information on medicinal plants, plant parts used, preparation methods, routes of administration, and knowledge transmission pathways was obtained. Quantitative ethnobotanical indices, including Frequency of Citation (FC), Relative Frequency of Citation (RFC), and Informant Consensus Factor (ICF), were calculated. Results: A total of 21 medicinal plant species were documented. The most frequently cited species were Moringa oleifera (FC = 9; RFC = 0.225), Vernonia amygdalina (FC = 7; RFC = 0.175), and both Cassia siberiana and Telfairia occidentalis (FC = 6; RFC = 0.150). Leaves were the most commonly utilized plant part (40.9%), and decoction was the predominant preparation method (76.2%), with oral administration accounting for 95.2% of use. The Informant Consensus Factor (ICF = 0.69) indicated a relatively high level of agreement among informants. Knowledge was primarily transmitted through apprenticeship and inherited family practices. Conclusion: Traditional medicinal plants remain an important component of diabetes management in Sierra Leone. The high level of consensus among informants and the repeated citation of specific plant species suggest structured and culturally validated therapeutic practices. The findings provide a foundation for future phytochemical and pharmacological investigations and highlight the need for documentation, preservation, and sustainable utilization of ethnobotanical knowledge.

Preeclampsia (PE), new onset hypertension after 20 weeks of gestation, affects 10% of all pregnancies in the U.S. and it is associated with progesterone deficiency, chronic inflammation, elevated angiotensin II type 1 receptor agonistic autoantibody (AT1-AA) and endothelial dysfunction. Progesterone, through its receptors, stimulates an anti- inflammatory protein called Progesterone Induced Blocking Factor (PIBF) which decreases during various pregnancy disorders. Therefore, this study was designed to test the hypothesis that a progestogen, in the form of 17-hydroxyprogesterone caproate, stimulates PIBF, lowers vasoactive mechanisms which reduces maternal blood pressure in women with early-onset preeclampsia (EOPE). PE women received 17-OHPC (250 mg, I.M.) and blood draws were collected before and after 17-OHPC supplementation. Placentas were collected at the delivery. 17-OHPC prolonged time of delivery beyond 72h on average and maternal blood pressure was significantly decreased in PE+17- OHPC. Progesterone and PIBF levels were reduced in PE group vs. NP group. Importantly, 17-OHPC increased PIBF and decreased vasoactive mechanisms and markers of inflammation. In conclusion, 17-OHPC or progesterone supplementation improves maternal outcomes in response to EOPE without causing further harm to the fetus.

Introduction: Conventional substance-level disproportionality analysis may miss safety patterns specific to a dose form, route, or intended site. More granular analyses are hindered by incomplete, inconsistent reporting of product information. The Pharmaceutical Product Identifier (PhPID), representing products by substance, strength, and dose form, may support more granular analyses. Objective: To explore the use of PhPID-like dose form information for site-specific disproportionality analysis in dexamethasone. Methods: We evaluated VigiBase reports (January 1, 2001 - December 31, 2024) for completeness of dose form and route data. We standardized dexamethasone entries to PhPID Level 3 standards, representing substance and administrable dose form. Through disproportionality analysis (Information Component, IC) we compared substance-level and site-specific results. Results: Among 56.4 million suspected/interacting drugs, dose form was reported in 47.7%, route in 69.4%. Among 109,248 dexamethasone entries, 703 dose form and 80 route variations were mapped to 53 and 44 standard codes respectively; about half could be mapped unambiguously. Site-specific analyses revealed biologically plausible patterns not apparent in substance-level analyses. Ocular use showed higher ICs for glaucoma and cataract, while systemic use showed higher IC for psychiatric and endocrine events (e.g., depression, agitation, Cushing's syndrome). IC time-trends suggested that some signals (e.g., cataract with Ocular use) could emerge earlier in site-specific analyses. Conclusion: More granular product information, aligned with PhPID, may improve signal detection and characterization of site-specific safety issues. These findings support granular identifiers in pharmacovigilance while highlighting the need for better capture and standardization of dose form and route of administration data.

BackgroundTuberculosis remains a leading global infectious killer, with BCG offering inconsistent adult protection and rising drug-resistant strains demanding novel vaccine strategies. We report the first multi-epitope vaccine construct simultaneously targeting three previously unexplored Mycobacterium tuberculosis virulence proteins; EccB3, MycP, and polyketide synthase which collectively govern nutrient acquisition, ESX secretion integrity, and innate immune evasion. MethodsUsing a reverse vaccinology pipeline, B-cell, CTL, and HTL epitopes were predicted, filtered for allergenicity, toxicity, and IFN-{gamma} induction, then assembled into an 823-residue chimeric construct incorporating beta-defensin and PADRE adjuvants with AAY/GPGPG linkers, covering [~]90% global HLA diversity. The construct underwent AlphaFold structure prediction, 3DRefine refinement, disulfide engineering, PROCHECK/ProSA validation, ClusPro 2.0 docking against TLR1/TLR2, and C-IMMSIM immune simulation. ResultsThe construct (82.3 kDa, instability index 32.48) showed strong structural quality (94.7% favoured Ramachandran residues), stable TLR1/TLR2 binding (weighted energy: -1,371.0 kcal/mol), and robust in silico immune responses and durable memory cell formation following booster simulation. ConclusionThis computationally validated construct represents a promising multi-target TB vaccine candidate warranting experimental advancement.

Objective: Photobiomodulation therapy has emerged as a promising modality to facilitate scar healing and pain management in dermatology and plastic surgery. However, its role in postoperative care following spine surgeries remains understudied. This double-blinded, placebo-controlled study aimed to investigate the effects of photobiomodulation in patients with chronic lower back pain undergoing lumbar decompression, with postoperative wound healing as the primary outcome and pain reduction and functional recovery as secondary outcomes. Methods: Patients were randomized to receive either active photobiomodulation braces (N=13) or placebo braces (N=12). Follow-up assessments were performed at 2, 4, 6, 8, and 12 weeks postoperatively. Outcomes included wound healing (Stony Brook Scar Evaluation Scale), back and leg pain (Visual Analog Scale), quality of life (EuroQol 5D), and functional status (Oswestry Disability Index). Results: Compared to the placebo group, the photobiomodulation treatment group had a 4.12-fold cumulative improvement in final scar scores, with significant between-group differences at postoperative weeks 6, 8, and 12 (p = 0.0062, 0.010, 0.042). Among patients with severe preoperative disability, treatment resulted in a 1.89-fold faster improvement in back pain (p=0.025) and a 1.80-fold faster improvement in ODI scores (p=0.025); and superior treatment effect on wound healing were again observed at weeks 6, 8, and 12. Among patients with poor initial scars, treatment led to a significantly better scar outcome than placebo at week 6 and a 1.94-fold faster EQ5D improvement (p=0.052), with significant gains observed as early as two weeks after surgery. There were no adverse events associated with photobiomodulation treatment. Conclusions: Photobiomodulation significantly promoted postoperative wound healing following lumbar decompression surgery, with therapeutic benefits preserved even in patients with poor baseline scar scores and functional impairment. This indicates that the efficacy of photobiomodulation is not limited by the initial scar condition or disability, supporting its broad clinical applicability. Additionally, patients with severe preoperative disability experienced greater benefits from photobiomodulation than placebo, including faster reduction in back pain and more rapid improvement in functional capacity, highlighting its role in postoperative pain management and rehabilitation. These therapeutic effects are likely mediated by photobiomodulation-induced reduction of inflammation and enhancement of tissue repair. Together, this study suggests that photobiomodulation can be a promising adjunct therapy to facilitate postoperative recovery in patients undergoing spine surgery.

Background: Short-chain fatty acids (SCFAs) are microbial metabolites with immunoregulatory properties. Human milk contains SCFAs which have been proposed as potential modulators of infant immune development. We aimed to examine associations between human milk SCFA concentrations and infant allergic disease outcomes in a high-risk cohort of infants of atopic mothers. Methods: SCFAs were measured by targeted liquid chromatography-mass spectrometry in human milk samples collected at 3 and 6 months postpartum from atopic mothers enrolled in the Infant Fish Oil Supplementation (IFOS) Study (n=147). Associations between milk SCFA concentrations and early childhood allergic disease outcomes (atopic dermatitis, food allergy, allergic rhinitis, and allergen sensitisation at 1 and 2-3 years) were examined using logistic regression. Results: Human milk SCFA concentrations were broadly stable between 3 and 6 months postpartum, except for acetate which was significantly elevated at 6 months. No significant associations were observed between human milk SCFA concentrations and any allergic disease outcome after correction for multiple comparisons (all p>0.05). Conclusions: Human milk SCFA concentrations are not associated with allergic disease outcomes up to 4 years of age. These findings suggest that oral SCFA exposure via human milk is insufficient to reduce infant allergy risk, and that gut SCFA production may be a more relevant target for future allergy prevention research.

"Mirror life", self-replicating organisms composed of non-natural-chirality biomacromolecules, presents a future threat with potentially global consequences. Consequently, there is strong agreement among experts that it should not be created. However, there is some disagreement over how effective existing medical countermeasures might prove against mirror bacteria in the event that they were created. Here, we leverage computational chemistry methods including docking and molecular dynamics to determine the likely binding efficacy of existing antibiotics against natural and mirror bacterial protein targets. We find that most existing antibiotics fail to bind to mirror bacterial protein targets, unlike their natural-chirality targets. This suggests altered binding of current medical countermeasures, which may impact the antimicrobial activity against mirror bacteria were the latter were created.

Background Liver transplantation is a complex surgical procedure featuring prolonged operative time and extensive surgical trauma, which results in a high anesthetic risk. Especially during anesthesia induction and the anhepatic-to-reperfusion phases, where marked hemodynamic fluctuations may readily lead to malignant cardiovascular events. Liver transplantation is associated with numerous postoperative complications, including pulmonary complications, postoperative delirium, acute kidney injury, and delayed emergence, all of which may adversely affect patient prognosis. Studies on remimazolam besylate (hereafter referred to as remimazolam) have suggested that it has minimal impact on patient hemodynamics. In theory, this renders remimazolam an ideal sedative agent for liver transplantation. This study aims to verify the hypothesis that the use of remimazolam during liver transplantation can reduce the incidence of postreperfusion syndrome (PRS). In addition, we further explored the effects of remimazolam on postoperative complications in liver transplant recipients, particularly focusing on perioperative liver and kidney function, pulmonary complications, and postoperative delirium. Methods This study is a prospective randomized controlled trial. 120 participants aged 18-60 years who are scheduled to undergo liver transplantation under general anesthesia will be enrolled. In the intervention group, remimazolam besylate will be used for anesthesia induction and maintenance at doses of 0.2-0.4 mg/kg and 1-3 mg/kg/h until the end of surgery. In the control group, propofol will be used for anesthesia induction and maintenance at doses of 1-2 mg/kg and 4-12 mg/kg/h until the end of surgery. In both groups, anesthetic drug doses or sevoflurane administration for intravenous-inhalational combined anesthesia will be adjusted based on vital signs and BIS values. All other anesthetic medications will be conducted according to the anesthetist's preference and remain consistent. Discussion This trial will investigate whether remimazolam besylate can be used during liver transplantation to reduce the incidence of postreperfusion syndrome. It will also examine whether the drug provides potential benefits regarding perioperative complications such as postoperative delirium and acute kidney injury. Trial Registration: Chinese Clinical Trial Registry,ChiCTR2500095774, registered on January 13, 2025 Keywords: liver transplantation, remimazolam, postreperfusion syndrome, complications

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

Recently, an observational study demonstrated that a lower fractional excretion of uric acid (FEUA) is significantly associated with a higher risk of kidney failure. This study aimed to assess the efficacy of switching from febuxostat to dotinurad, which increases FEUA, in patients with chronic kidney disease (CKD) and hyperuricemia (HUA).This was a non-randomized, open-label, single-center, prospective, single-arm study involving 60 patients with CKD and HUA who received febuxostat. Participants first underwent a 3-month observation period, followed by a 3-month intervention period, during which treatment was switched from febuxostat to dotinurad. The primary outcomes were changes from baseline to 3-months after switching in the estimated glomerular filtration rate (eGFR) calculated from serum creatinine (eGFRcreat) and serum cystatin C (eGFRcys), as well as the serum uric acid levels. The secondary outcome was defined as the correlation between{Delta}FEUA and the changes in both eGFRcreat({Delta}eGFRcreat) and eGFRcys({Delta}eGFRcys), respectively. During the observation period, mean eGFRcreat decreased significantly. The baseline eGFRcreat (mL/min/1.73 m{superscript 2}) was 36.0 {+/-} 15.2, and the serum urate level (mg/dL) was 5.5 {+/-} 1.2. During the intervention period, eGFRcreat increased in contrast to the significant decline observed in eGFRcys. After 3 months of switching to dotinurad, the mean serum UA levels increased significantly from 5.5 {+/-} 1.2 to 6.1 {+/-} 1.4 mg/dL, despite a significant elevation in FEUA. Both {Delta}eGFRcreat and {Delta}eGFRcys after switching to dotinurad were positively correlated with {Delta}FEUA. Switching from febuxostat to dotinurad resulted in discrepant changes in eGFRcreat and eGFRcys, suggesting that renal function should be assessed carefully after switch. Additionally, the risk of elevated serum UA levels should be considered when switching from febuxostat to dotinurad in patients with CKD.

Methods INCA is a prospective, single-center cohort study with nationwide recruitment. Participation is open to adult patients and informal caregivers who provide paid informal care through home care agencies (Spitex organizations) in Switzerland. Eligible participants are enrolled consecutively. The cohorts primary outcome is health-related quality of life of patients, assessed monthly through patient-reported outcome measures. Secondary outcomes include home care needs, including the overall health and well-being of patients (measured semi-annually), the type, amount, and quality of care (recorded daily), and caregiver burden and resilience (measured quarterly). Additional analysis will include structured medical data, extracted from patient-provided documents using Optical Character Recognition (OCR) technology and analysed using Large Language Models (LLM). Results Since recruiting started in July 2025, the cohort has enrolled 855 patients and 851 caregivers. Among patients, 53% are female, with a median age of 73 years. Caregivers are predominantly female (72%) with a median age of 56 years. Most patients experience impairments in physical functioning and participation in social roles. Among them, 85% require less than two hours of care per day, though care needs vary considerably. This is further reflected in the multi-attribute utility, where the overall PROMIS-Preference (PROPr) score is very low for most patients, with a median of 0.102, indicating a substantial need for medical assistance and care. With a median of 9 comorbidities, health-related quality of life is overall low for most patients. Cardiovascular and endocrine & metabolic diseases are amongst the most prevalent, affecting 69% and 65% of patients with available diagnoses (n=771). Certain diagnostic pairs occur more frequently than expected by chance, suggesting underlying links between disease categories. Conclusions INCA responds to a growing policy need for robust evidence on how new models of informal care are associated with the health and well-being of patients and their caregivers. Its longitudinal design, combining patient- and caregiver-reported data with medical records and innovative data extraction methods, will lay the groundwork for a better understanding of new informal care models in real-world settings. INCAs findings are expected to have significant policy relevance and contribute to evidence-based policies on long-term care at home in Switzerland.

This qualitative study evaluated the Life Skills (HpV) training strategy at a public university in Colombia in 2024, analyzing its impact on students positive mental health and psychosocial competencies. The mixed-methods research employed semi-structured interviews with faculty and three student focus groups, using thematic analysis to categorize strengths, weaknesses, and perceived changes. Results highlighted curricular coherence, academic freedom, and participatory methodology as key aspects, fostering self-awareness, emotional management, and the building of support networks. Students reported improvements in well-being, stress management, and academic performance, though challenges such as initial resistance to emotional content and student diversity were identified. The conclusions underscore the value of experiential courses in university education, promoting horizontal relationships and safe spaces for collective reflection. Future studies are recommended to expand participant diversity and incorporate quantitative data triangulation to further explore the interventions effects.

Background Having achieved the UNAIDS 95-95-95 targets, Eswatini faces a growing burden of non-communicable diseases which are major contributors to morbidity and mortality. Hypertension-associated cardiovascular disease (CVD) is rising among people living with HIV (PLHIV) as survival improves and metabolic risks - including those linked to dolutegravir (DTG) - increase. We projected CVD burden among PLHIV and HIV-negative adults (PLWHIV) through 2045 to inform integrated HIV-CVD planning. Methods EMOD-HIV, an agent-based model calibrated to Eswatini's epidemic, generated HIV prevalence trajectories. These were combined with age-standardized Global Burden of Disease CVD estimates and published relative risks (RRs) to produce HIV-stratified CVD projections. CVD burden trajectories were then projected through 2045 using a logistic generalized additive model with Monte Carlo uncertainty quantification. Five scenarios were evaluated to assess how different assumptions about RR of CVD among PLHIV versus PLWHIV affect projected burden: (1) CVD prevalence under a constant RR; (2) CVD mortality under a constant RR; (3) HTN-attributable CVD mortality under a constant RR; (4) HTN-attributable CVD mortality under a post-DTG RR increase following Eswatinis 2021 dolutegravir rollout; and (5) HTN-attributable CVD mortality under a gradual RR increase from 2010-2045 reflecting cumulative metabolic and demographic shifts. Results PLHIV consistently exhibited higher CVD burden than HIV-negative adults. Scenario 1: CVD prevalence was 11.0% (95% UI: 9.0-13.5%) among PLHIV versus 6.8% (6.0-7.8%) in 2025, stable through 2045. Scenario 2: CVD mortality rate was 0.60% (0.47-0.76%) versus 0.37% (0.31-0.45%) in 2025, declining modestly through 2045 with consistent excess. Scenario 3: HTN-attributable mortality was 73% (70-76%) versus 64% (61-66%) in women and 61% (58-64%) versus 58% (55-60%) in men, stable through 2045. Scenario 4: Following DTG rollout, mortality rose from 73% to 85% in women and 61% to 70% in men by 2022, remaining stable thereafter. Scenario 5: By 2045, mortality reached 85% (82-88%) in women and 67% (64-70%) in men with HIV, versus 60% (57 - 63%) and 55% (53 - 57%) in HIV-negative adults. Conclusions While excess CVD burden among PLHIV is projected to persist even under stable risk conditions, ART-related metabolic trajectories - particularly those linked to DTG - may drive substantial widening of this gap through 2045. HTN-attributable CVD mortality is particularly elevated among women with HIV. Strengthening integrated HIV-NCD services, including blood pressure screening, risk-based therapy, and sex-specific DTG counseling, will be essential to sustain long-term health gains.

Internal-tandem-duplication of the receptor tyrosine kinase FLT3 (FLT3-ITD) generates ligand-independent signaling and is highly recurrent in acute myeloid leukemias (AMLs). One way signaling pathways can quickly influence cell fates is by phosphorylating key fate-determining proteins to trigger their proteolysis. We investigated the master transcription factor (MTF) driver of granulo-monocytic lineage-fates, CEBPA, for regulation by this mechanism because we found high CEBPA mRNA but little CEBPA protein in FLT3-ITD versus FLT3-wildtype AML cells, and inhibiting FLT3-ITD signaling with tyrosine kinase inhibitors (TKI) rapidly rescued CEBPA protein. Mass spectrometry analyses of CEBPA and its interactome demonstrated prominent interactions with major ubiquitin-proteosome pathway (UPP) components UHRF1 and USP7. TKI treatments decreased CEBPA and USP7 phosphorylations at serine 21 and serine 18 respectively alongside shifts in CEBPA interactions from degradative ubiquitin-ligase UHRF1 toward protective deubiquitinase USP7. The rescued CEBPA activated granulocytic-differentiation. Supporting that the serine-phosphorylations were phospho-degrons, UPP-inhibitors (bortezomib, MG132) increased phosphorylated and total CEBPA and USP7. The MTF regulator of apoptosis p53 is a known USP7 client, therefore, we also evaluated p53 status: TKIs and UPP-inhibitors stabilized USP7 and p53, triggering apoptosis in addition to granulocytic-differentiation specifically in FLT3-ITD but not FLT3-wildtype AML cells. UPP-inhibitors produced these consequences in TKI-resistant FLT3-ITD AML cells also. These data predicted genetic loss-of-function to CEBPA or TP53 is redundant in the FLT3-ITD context, borne out by mutual exclusivity of the mutations in clinical series. In summary, FLT3-ITD signals for CEBPA and p53 proteolysis to block lineage-maturation and apoptosis, positioning UPP-inhibitors as therapeutic candidates acting downstream of TKIs. KEY POINTSO_LIThe oncoprotein kinase FLT3-ITD signals for CEBPA and p53 proteolysis and hence suppresses lineage-differentiation and apoptosis C_LIO_LIProteosome-inhibitors are candidate remedies to restore CEBPA and p53, acting downstream of presently used FLT3-ITD kinase inhibitors C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/738455v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@6ae211org.highwire.dtl.DTLVardef@12003bforg.highwire.dtl.DTLVardef@d62eb9org.highwire.dtl.DTLVardef@1958693_HPS_FORMAT_FIGEXP M_FIG C_FIG

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this "OTTR-CLASH" method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/738487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13bc276org.highwire.dtl.DTLVardef@5beb41org.highwire.dtl.DTLVardef@b204e5org.highwire.dtl.DTLVardef@15f747d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Natural product synthesis by non-ribosomal peptide synthetases (NRPS) is greatly defined by the substrate selectivity of the adenylation (A) domains. Previous assays for specificity determination were mainly performed in vitro and were requiring protein purification. In this work, we developed - based on NRPS engineering - a novel in vivo assay suitable for high-throughput application named ASCR (A domain screening). Using the recently described XUT fusion sites, A domains and their upstream condensation domains were assembled as di-domains to characterized NRPS model system, which allowed detection of defined tripeptide products via mass spectrometry directly after cell culture extraction. We evaluated the assay by screening in total 54 A domains from five known and seven uncharacterized NRPS, covering a broad range organism taxonomy and GC content of the investigated NRPS-encoding genes. Additionally, we applied the assay to elucidate and confirm the structures of novel cyclic pentapeptides derived from three novel NRPS from Photorhabdus temperata K122.

Prenatal alcohol exposure (PAE) is a major cause of neurodevelopmental disorders, yet most children are diagnosed late or misdiagnosed. Neuroplacentology suggest that placental factors released into maternal and/or umbilical cord blood contribute to fetal brain development. Consistently, a preclinical inter-organ transcriptomic database revealed that PAE disrupts the expression ratio of angiogenic and inflammatory factors suggesting an angio-inflammatory response. This study aimed i) to assay, by multiplex immunoassay, angiogenic and inflammatory factors in maternal and umbilical cord blood from alcohol-consuming women and ii) to perform a maternofetal analysis according to neonatal sex. Afterwards, dysregulated factors from mothers who gave birth to females or males were submitted to STRING and ShinyGO analyses. Results showed that PAE differently altered the distribution profiles of dysregulated angiogenic and inflammatory factors in maternal and umbilical cord blood. Moreover, sex-specific differences were observed, with 36% of dysregulated proteins specific to males, 48% to females, and 16% common to both. STRING analysis revealed robust functional protein-protein interactions linking together inflammatory and angiogenic clusters while the ShinyGO analysis identified enriched pathways related to vascular shear stress. These findings provide the first maternofetal analysis of combined angiogenic and inflammatory factors from alcohol-consuming mothers.