Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer.

Technologically critical rare-earth elements are notoriously difficult to separate, owing to their subtle differences in ionic radius and coordination number1-3. The natural lanthanide-binding protein lanmodulin (LanM)4,5 is a sustainable alternative to conventional solvent-extraction-based separation6. Here we characterize a new LanM, from Hansschlegelia quercus (Hans-LanM), with an oligomeric state sensitive to rare-earth ionic radius, the lanthanum(III)-induced dimer being >100-fold tighter than the dysprosium(III)-induced dimer. X-ray crystal structures illustrate how picometre-scale differences in radius between lanthanum(III) and dysprosium(III) are propagated to Hans-LanM's quaternary structure through a carboxylate shift that rearranges a second-sphere hydrogen-bonding network. Comparison to the prototypal LanM from Methylorubrum extorquens reveals distinct metal coordination strategies, rationalizing Hans-LanM's greater selectivity within the rare-earth elements. Finally, structure-guided mutagenesis of a key residue at the Hans-LanM dimer interface modulates dimerization in solution and enables single-stage, column-based separation of a neodymium(III)/dysprosium(III) mixture to >98% individual element purities. This work showcases the natural diversity of selective lanthanide recognition motifs, and it reveals rare-earth-sensitive dimerization as a biological principle by which to tune the performance of biomolecule-based separation processes.

Phase separation enhances probability of receptor signalling and drug targeting.

The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a 'numbers game', where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid-liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.

Excess dietary sugar impairs Drosophila adult stem cells via elevated reactive oxygen species-induced JNK signaling.

High-sugar diets (HSDs) often lead to obesity and type 2 diabetes, both metabolic syndromes associated with stem cell dysfunction. However, it is unclear whether excess dietary sugar affects stem cells. Here, we report that HSD impairs stem cell function in the intestine and ovaries of female Drosophila prior to the onset of insulin resistance, a hallmark of type 2 diabetes. Although 1 week of HSD leads to obesity, impaired oogenesis and altered lipid metabolism, insulin resistance does not occur. HSD increases glucose uptake by germline stem cells (GSCs) and triggers reactive oxygen species-induced JNK signaling, which reduces GSC proliferation. Removal of excess sugar from the diet reverses these HSD-induced phenomena. A similar phenomenon is found in intestinal stem cells (ISCs), except that HSD disrupts ISC maintenance and differentiation. Interestingly, tumor-like GSCs and ISCs are less responsive to HSD, which may be because of their dependence on glycolytic metabolism and high energy demand, respectively. This study suggests that excess dietary sugar induces oxidative stress and damages stem cells before insulin resistance develops, a mechanism that may also occur in higher organisms.

Inhibition of basal FGF receptor signaling by dimeric Grb2.

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.

A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly.

RNA flow between organisms has been documented within and among different kingdoms of life. Recently, we demonstrated horizontal RNA transfer between honeybees involving secretion and ingestion of worker and royal jellies. However, how the jelly facilitates transfer of RNA is still unknown. Here, we show that worker and royal jellies harbor robust RNA-binding activity. We report that a highly abundant jelly component, major royal jelly protein 3 (MRJP-3), acts as an extracellular non-sequence-specific RNA-aggregating factor. Multivalent RNA binding stimulates higher-order assembly of MRJP-3 into extracellular ribonucleoprotein granules that protect RNA from degradation and enhance RNA bioavailability. These findings reveal that honeybees have evolved a secreted dietary RNA-binding factor to concentrate, stabilize, and share RNA among individuals. Our work identifies high-order ribonucleoprotein assemblies with functions outside cells and organisms.

α-Ketoglutarate plays an inflammatory inhibitory role by regulating scavenger receptor class a expression through N6-methyladenine methylation during sepsis.

Sepsis arises from an uncontrolled inflammatory response triggered by infection or stress, accompanied by alteration in cellular energy metabolism, and a strong correlation exists between these factors. Alpha-ketoglutarate (α-KG), an intermediate product of the TCA cycle, has the potential to modulate the inflammatory response and is considered a crucial link between energy metabolism and inflammation. The scavenger receptor (SR-A5), a significant pattern recognition receptor, assumes a vital function in anti-inflammatory reactions. In the current investigation, we have successfully illustrated the ability of α-KG to mitigate inflammatory factors in the serum of septic mice and ameliorate tissue damage. Additionally, α-KG has been shown to modulate metabolic reprogramming and macrophage polarization. Moreover, our findings indicate that the regulatory influence of α-KG on sepsis is mediated through SR-A5. We also elucidated the mechanism by which α-KG regulates SR-A5 expression and found that α-KG reduced the N6-methyladenosine level of macrophages by up-regulating the m6A demethylase ALKBH5. α-KG plays a crucial role in inhibiting inflammation by regulating SR-A5 expression through m6A demethylation during sepsis. The outcomes of this research provide valuable insights into the relationship between energy metabolism and inflammation regulation, as well as the underlying molecular regulatory mechanism.

An Unusual Ferryl Intermediate and Its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO.

N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Deleterious genetic changes in AGTPBP1 result in teratozoospermia with sperm head and flagella defects.

Approximately 10%-15% of couples worldwide are infertile, and male factors account for approximately half of these cases. Teratozoospermia is a major cause of male infertility. Although various mutations have been identified in teratozoospermia, these can vary among ethnic groups. In this study, we performed whole-exome sequencing to identify genetic changes potentially causative of teratozoospermia. Out of seven genes identified, one, ATP/GTP Binding Protein 1 (AGTPBP1), was characterized, and three missense changes were identified in two patients (Affected A: p.Glu423Asp and p.Pro631Leu; Affected B: p.Arg811His). In those two cases, severe sperm head and tail defects were observed. Moreover, AGTPBP1 localization showed a fragmented pattern compared to control participants, with specific localization in the neck and annulus regions. Using murine models, we found that AGTPBP1 is localized in the manchette structure, which is essential for sperm structure formation. Additionally, in Agtpbp1-null mice, we observed sperm head and tail defects similar to those in sperm from AGTPBP1-mutated cases, along with abnormal polyglutamylation tubulin and decreasing △-2 tubulin levels. In this study, we established a link between genetic changes in AGTPBP1 and human teratozoospermia for the first time and identified the role of AGTPBP1 in deglutamination, which is crucial for sperm formation.

Trends of pre-treatment drug resistance in antiretroviral-naïve people with HIV-1 in the era of second-generation integrase strand-transfer inhibitors in Taiwan.

BACKGROUND: Monitoring the trends of pre-treatment drug resistance (PDR) and resistance-associated mutations (RAMs) among antiretroviral-naïve people with HIV (PWH) is important for the implementation of HIV treatment and control programmes. We analysed the trends of HIV-1 PDR after the introduction of second-generation integrase strand-transfer inhibitors (INSTIs) in 2016 in Taiwan, when single-tablet regimens of non-nucleoside reverse-transcriptase inhibitor (NNRTI-) and INSTI-based antiretroviral therapy became the preferred treatments. MATERIALS AND METHODS: In this multicentre study, we included newly diagnosed, antiretroviral-naïve PWH who underwent tests for RAMs between 2016 and 2022. Pre-treatment genotypic resistance testing was performed, along with HIV-1 subtyping and determinations of plasma HIV RNA load and CD4 lymphocyte counts. RAMs were analysed using the Stanford University HIV Drug Resistance Database and only RAMs conferring at least low-level resistance were included. RESULTS: From 2016 to 2022, pre-treatment blood samples from 3001 newly diagnosed PWH, which constituted 24.3% of newly diagnosed PWH in Taiwan during the study period, were tested. Of the PWH with analysable gene sequences, the HIV-1 PDR prevalence to NNRTIs, nucleoside reverse-transcriptase inhibitors (NRTIs), first- and second-generation INSTIs and PIs was 10.0%, 2.1%, 2.5%, 0.6% and 0.4%, respectively. While the trends of PDR remained stable for NRTIs, INSTIs and PIs, there was a significantly increasing trend of PDR to NNRTIs from 6.0% in 2016% to 13.1% in 2022 (P = 0.001). CONCLUSIONS: After the introduction of second-generation INSTIs in Taiwan, the trends of HIV-1 PDR to NRTIs and INSTIs remained low. Furthermore, there was no significant decrease of the prevalence of PDR toward NNRTIs between 2016 and 2022.

A phase 1 trial of the MEK inhibitor selumetinib in combination with pembrolizumab for advanced or metastatic solid tumors.

MEK inhibitors have immunomodulatory activity and potential for synergistic activity when combined with PD-1 inhibitors. We evaluated selumetinib (inhibitor of MEK1/2) plus pembrolizumab (anti‒PD-1 antibody) in patients with advanced/metastatic solid tumors. In this phase 1b study, adults with previously treated advanced/metastatic solid tumors received pembrolizumab 200 mg intravenously every 3 weeks plus selumetinib on days 1‒14 per 3-week cycle (2 weeks on/1 week off); selumetinib dosing began at 50 mg orally twice daily with escalation in 25 mg increments for ≤ 35 cycles. Primary endpoints were dose-limiting toxicities (DLTs), adverse events (AEs), and treatment discontinuations due to AEs. Thirty-two patients were enrolled. Dose escalation was completed up to selumetinib 125 mg twice daily. The target DLT rate of 30% was not reached at any dose level. In the selumetinib 100 mg group, 2/11 patients (18.2%) experienced DLTs (n = 1 grade 3 diarrhea, n = 1 grade 3 fatigue). In the selumetinib 125 mg group, 3/14 (21.4%) experienced DLTs (n = 1 grade 2 retinal detachment, n = 1 grade 3 retinopathy, n = 1 grade 3 stomatitis). Dose-related changes in pharmacokinetic exposures were observed for selumetinib and N-desmethyl selumetinib up to 100 mg (saturation at 125 mg). Two patients achieved partial responses (1 each with selumetinib 75 mg and 125 mg) for an objective response rate of 6%. The study was stopped early because of insufficient efficacy. Although the target DLT rate was not reached at any dose level and no new safety signals were identified, selumetinib plus pembrolizumab had limited antitumor activity in this population. Trial registration: ClinicalTrials.gov , NCT03833427.

Increased di-(2-ethylhexyl) phthalate exposure poses a differential risk for adult asthma clusters.

BACKGROUND: DEHP, a common plasticizer known for its hormone-disrupting properties, has been associated with asthma. However, a significant proportion of adult asthma cases are "non-atopic", lacking a clear etiology. METHODS: In a case-control study conducted between 2011 and 2015, 365 individuals with current asthma and 235 healthy controls from Kaohsiung City were enrolled. The control group comprised individuals without asthma, Type 2 Diabetes Mellitus (T2DM), hypertension, or other respiratory/allergic conditions. The study leveraged asthma clusters (Clusters A to F) established in a prior investigation. Analysis involved the examination of urinary DEHP metabolites (MEHP and MEHHP), along with the assessment of oxidative stress, sphingolipid metabolites, and inflammatory biomarkers. Statistical analyses encompassed Spearman's rank correlation coefficients, multiple logistic regression, and multinomial logistic regression. RESULTS: Asthma clusters (E, D, C, F, A) exhibited significantly higher ORs of MEHHP exposures compared to the control group. When considering asthma-related comorbidities (T2DM, hypertension, or both), patients without comorbidities demonstrated significantly higher ORs of the sum of primary and secondary metabolites (MEHP + MEHHP) and MEHHP compared to those with asthma comorbidities. A consistent positive correlation between urinary HEL and DEHP metabolites was observed, but a consistent negative correlation between DEHP metabolites and selected cytokines was identified. CONCLUSION: The current study reveals a heightened risk of MEHHP and MEHP + MEHHP exposure in specific asthma subgroups, emphasizing its complex relationship with asthma. The observed negative correlation with cytokines suggests a new avenue for research, warranting robust evidence from epidemiological and animal studies.

The difference between dacomitinib and afatinib in effectiveness and safety in first-line treatment of patients with advanced EGFR-mutant non-small cell lung cancer: a real-world observational study.

OBJECTIVES: The irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) afatinib and dacomitinib are approved for first-line treatment of EGFR mutation-positive non-small cell lung cancer (NSCLC). We aimed to compare the efficacy and safety of afatinib and dacomitinib in this setting. MATERIALS AND METHODS: Between September 2020 and March 2023, we retrospectively recruited patients diagnosed with advanced-stage EGFR-mutant NSCLC who were treated with first-line irreversible EGFR-TKIs. The enrolled patients were assigned to two groups based on whether they received afatinib or dacomitinib. RESULTS: A total of 101 patients were enrolled in the study (70 to afatinib and 31 to dacomitinib). The partial response rates (PR) for first-line treatment with afatinib and dacomitinib were 85.7 and 80.6% (p = 0.522). The median progression-free survival (PFS) (18.9 vs. 16.3 months, p = 0.975) and time to treatment failure (TTF) (22.7 vs. 15.9 months, p = 0.324) in patients with afatinib and dacomitinib treatment were similar. There was no significant difference observed in the median PFS (16.1 vs. 18.9 months, p = 0.361) and TTF (32.5 vs. 19.6 months, p = 0.182) between patients receiving the standard dose and those receiving the reduced dose. In terms of side effects, the incidence of diarrhea was higher in the afatinib group (75.8% vs. 35.5%, p < 0.001), while the incidence of paronychia was higher in the dacomitinib group (58.1% vs. 31.4%, p = 0.004). The PFS (17.6 vs. 24.9 months, p = 0.663) and TTF (21.3 vs. 25.1 months, p = 0.152) were similar between patients younger than 75 years and those older than 75 years. CONCLUSION: This study showed that afatinib and dacomitinib had similar effectiveness and safety profiles. However, they have slightly different side effects. Afatinib and dacomitinib can be safely administered to patients across different age groups with appropriate dose reductions.

The impact of squamous cell transformation on the prognosis of patients treated with radical nephroureterectomy.

BACKGROUND: Limited information is available for guiding the management of upper urinary tract (UUT) urothelial carcinoma with squamous differentiation (UC-SqD). We did not even know about the difference between pure urothelial carcinoma (UC) and UC-SqD in the UUT regardless of treatment policy and prognosis. Instead of direct comparisons against each other, we included the third UUT malignancy, squamous cell carcinoma (SCC). This three-way-race model allows us to more clearly demonstrate the impact of squamous cell transformation on patient outcomes in UUT malignancy. METHODS: We retrospectively analysed 327 patients with UC, UC-SqD, or SCC who underwent radical nephroureterectomy with bladder cuff excision (RNU) at Taichung Veterans General Hospital, Taichung, Taiwan, between January 2006 and December 2013. A Kaplan-Meier survival analysis was used to evaluate the relationship between patient outcomes and histology. Multivariate Cox proportional hazards modelling was also used to predict patient prognoses. RESULTS: The five-year postoperative cancer-specific survival (CSS) rates were 83.6% (UC), 74.4% (UC-SqD), and 55.6% (SCC), and the 5-year recurrence-free survival (RFS) rates were 87.7% (UC), 61.5% (UC-SqD), and 51.9% (SCC). UC patients had significantly better 5-year RFS than UC-SqD and SCC patients (P = 0.001 and P < 0.0001, respectively). Patients with pure UC had significantly better 5-year CSS than SCC patients (P = 0.0045). SCC or UC-SqD did not independently predict disease-specific mortality (HR 0.999, p = 0.999; HR 0.775, p = 0.632, respectively) or disease recurrence compared to pure UC (HR 2.934, p = 0.239; HR 1.422, p = 0.525, respectively). Age, lymphovascular invasion (LVI), and lymph node (LN) status independently predicted CSS, while pathological tumour stage, LN status, and LVI predicted RFS. CONCLUSIONS: SCC and UC-SqD are not independent predictors of survival outcomes in patients with UUT tumours. However, they are associated with other worse prognostic factors. Hence, different treatments are needed for these two conditions, especially for SCC.

Early-Life Social Determinants of SCA6 Age at Onset, Severity, and Progression.

SCA6 patients with the same size CAG repeat allele can vary significantly in age at onset (AAO) and clinical progression. The specific external factors affecting SCA6 have yet to be investigated. We assessed the effect of early life events on AAO, severity, and progression in SCA6 patients using a social determinant of health approach. We performed a survey of biological and social factors in SCA6 patients enrolled in the SCA6 Network at the University of Chicago. AAO of ataxia symptoms and patient-reported outcome measure (PROM) of ataxia were used as primary outcome measures. Least absolute shrinkage and selection operation (LASSO) regressions were used to identify which early life factors are predictive of SCA6 AAO, severity, and progression. Multiple linear regression models were then used to assess the degree to which these determinants influence SCA6 health outcomes. A total of 105 participants with genetically confirmed SCA6 completed the assessments. SCA6 participants with maternal difficulty during pregnancy, active participation in school sports, and/or longer CAG repeats were determined to have earlier AAO. We found a 13.44-year earlier AAO for those with maternal difficulty in pregnancy than those without (p = 0.008) and a 12.31-year earlier AAO for those active in school sports than those who were not (p < 0.001). Higher education attainment was associated with decreased SCA6 severity and slower progression. Early life biological and social factors can have a strong influence on the SCA6 disease course, indicating that non-genetic factors can contribute significantly to SCA6 health outcomes.

Consensus Paper: Cerebellum and Reward.

Cerebellum is a key-structure for the modulation of motor, cognitive, social and affective functions, contributing to automatic behaviours through interactions with the cerebral cortex, basal ganglia and spinal cord. The predictive mechanisms used by the cerebellum cover not only sensorimotor functions but also reward-related tasks. Cerebellar circuits appear to encode temporal difference error and reward prediction error. From a chemical standpoint, cerebellar catecholamines modulate the rate of cerebellar-based cognitive learning, and mediate cerebellar contributions during complex behaviours. Reward processing and its associated emotions are tuned by the cerebellum which operates as a controller of adaptive homeostatic processes based on interoceptive and exteroceptive inputs. Lobules VI-VII/areas of the vermis are candidate regions for the cortico-subcortical signaling pathways associated with loss aversion and reward sensitivity, together with other nodes of the limbic circuitry. There is growing evidence that the cerebellum works as a hub of regional dysconnectivity across all mood states and that mental disorders involve the cerebellar circuitry, including mood and addiction disorders, and impaired eating behaviors where the cerebellum might be involved in longer time scales of prediction as compared to motor operations. Cerebellar patients exhibit aberrant social behaviour, showing aberrant impulsivity/compulsivity. The cerebellum is a master-piece of reward mechanisms, together with the striatum, ventral tegmental area (VTA) and prefrontal cortex (PFC). Critically, studies on reward processing reinforce our view that a fundamental role of the cerebellum is to construct internal models, perform predictions on the impact of future behaviour and compare what is predicted and what actually occurs.

Cognitive, Emotional, and Other Non-motor Symptoms of Spinocerebellar Ataxias.

PURPOSE OF REVIEW: Spinocerebellar ataxias (SCAs) are autosomal dominant degenerative syndromes that present with ataxia and brain stem abnormalities. This review describes the cognitive and behavioral symptoms of SCAs in the context of recent knowledge of the role of the cerebellum in higher intellectual function. RECENT FINDINGS: Recent studies suggest that patients with spinocerebellar ataxia can display cognitive deficits even early in the disease. These have been given the term cerebellar cognitive affective syndrome (CCAS). CCAS can be tracked using newly developed rating scales. In addition, patients with spinocerebellar ataxia also display impulsive and compulsive behavior, depression, anxiety, fatigue, and sleep disturbances. This review stresses the importance of recognizing non-motor symptoms in SCAs. There is a pressing need for novel therapeutic interventions to address these symptoms given their deleterious impact on patients' quality of life.

Promoting Growth in Behavioral Neurology: A Path Forward.

Behavioral neurology & neuropsychiatry (BNNP) is a field that seeks to understand brain-behavior relationships, including fundamental brain organization principles and the many ways that brain structures and connectivity can be disrupted, leading to abnormalities of behavior, cognition, emotion, perception, and social cognition. In North America, BNNP has existed as an integrated subspecialty through the United Council for Neurologic Subspecialties since 2006. Nonetheless, the number of behavioral neurologists across academic medical centers and community settings is not keeping pace with increasing clinical and research demand. In this commentary, we provide a brief history of BNNP followed by an outline of the current challenges and opportunities for BNNP from the behavioral neurologist's perspective across clinical, research, and educational spheres. We provide a practical guide for promoting BNNP and addressing the shortage of behavioral neurologists to facilitate the continued growth and development of the subspecialty. We also urge a greater commitment to recruit trainees from diverse backgrounds so as to dismantle persistent obstacles that hinder inclusivity in BNNP-efforts that will further enhance the growth and impact of the subspecialty. With rapidly expanding diagnostic and therapeutic approaches across a range of conditions at the intersection of neurology and psychiatry, BNNP is well positioned to attract new trainees and expand its reach across clinical, research, and educational activities.

Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism.

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson-Crick base pair at the two drug-binding interfaces can result in a single-stranded 'chair-shaped' DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a 'curved-shaped' duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs' potential synergetic use.

Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the ∼4-ps population, P+HA- forms through a two-step process, P*→ P+BA-→ P+HA-, while in the ∼20-ps population, it forms via a one-step P* → P+HA- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.