A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease.

Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

Multi-omics analyses provide insights into the genomic basis of differentiation among four sweet osmanthus groups.

Sweet osmanthus (Osmanthus fragrans) is famous in China for its flowers and contains four groups: Albus, Luteus, Aurantiacus, and Asiaticus. Understanding the relationships among these groups and the genetic mechanisms of flower color and aroma biosynthesis are of tremendous interest. In this study, we sequenced representative varieties from two of the four sweet osmanthus groups. Multi-omic and phylogenetic analyses of varieties from each of the four groups showed that Asiaticus split first within the species, followed by Aurantiacus and the sister groups Albus and Luteus. We show that the difference in flower color between Aurantiacus and the other three groups was caused by a 4-bp deletion in the promoter region of carotenoid cleavage dioxygenase 4 (OfCCD4) that leads to expression decrease. In addition, we identified 44 gene pairs exhibiting significant structural differences between the multi-seasonal flowering variety 'Rixianggui' in the Asiaticus group and other autumn flowering varieties. Through correlation analysis between intermediate products of aromatic components and gene expression, we identified eight genes associated with the linalool, α- and ß-ionone biosynthesis pathways. Overall, our study offers valuable genetic resources for sweet osmanthus, while also providing genetic clues for improving the flower color and multi-season flowering of osmanthus and other flowers.

Differential mRNA profiles reveal the potential roles of genes involved in lactate stimulation in mouse macrophages.

Lactate is a glycolysis end product, and its levels are markedly associated with disease severity, morbidity, and mortality in sepsis. It modulates key functions of immune cells, including macrophages. In this investigation, transcriptomic analysis was performed using lactic acid, sodium lactate, and hydrochloric acid-stimulated mouse bone marrow-derived macrophages (iBMDM), respectively, to identify lactate-associated signaling pathways. After 24 h of stimulation, 896 differentially expressed genes (DEG) indicated were up-regulation, whereas 792 were down-regulated in the lactic acid group, in the sodium lactate group, 128 DEG were up-regulated, and 41 were down-regulated, and in the hydrochloric acid group, 499 DEG were up-regulated, and 285 were down-regulated. Subsequently, clinical samples were used to further verify the eight genes with significant differences, among which Tssk6, Ypel4, Elovl3, Trp53inp1, and Cfp were differentially expressed in patients with high lactic acid, indicating their possible involvement in lactic acid-induced inflammation and various physiological diseases caused by sepsis. However, elongation of very long chain fatty acids protein 3 (Elovl3) was negatively correlated with lactic acid content in patients. The results of this study provide a necessary reference for better understanding the transcriptomic changes caused by lactic acid and explain the potential role of high lactic acid in the regulation of macrophages in sepsis.

Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein Stability and Unfolding Dynamics.

How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.

Panda-UV Unlocks Deeper Protein Characterization with Internal Fragments in Ultraviolet Photodissociation Mass Spectrometry.

Ultraviolet photodissociation (UVPD) mass spectrometry unlocks insights into the protein structure and sequence through fragmentation patterns. While N- and C-terminal fragments are traditionally relied upon, this work highlights the critical role of internal fragments in achieving near-complete sequencing of protein. Previous limitations of internal fragment utilization, owing to their abundance and potential for random matching, are addressed here with the development of Panda-UV, a novel software tool combining spectral calibration, and Pearson correlation coefficient scoring for confident fragment assignment. Panda-UV showcases its power through comprehensive benchmarks on three model proteins. The inclusion of internal fragments boosts identified fragment numbers by 26% and enhances average protein sequence coverage to a remarkable 93% for intact proteins, unlocking the hidden region of the largest protein carbonic anhydrase II in model proteins. Notably, an average of 65% of internal fragments can be identified in multiple replicates, demonstrating the high confidence of the fragments Panda-UV provided. Finally, the sequence coverages of mAb subunits can be increased up to 86% and the complementary determining regions (CDRs) are nearly completely sequenced in a single experiment. The source codes of Panda-UV are available at https://github.com/PHOENIXcenter/Panda-UV.

Cyano-Rich g-C3 N4 in Photochemistry: Design, Applications, and Prospects.

Cyano-rich g-C3 N4 materials are widely used in various fields of photochemistry due to the very powerful electron-absorbing ability and electron storage function of cyano, as well as its advantages in improving light absorption, adjusting the energy band structure, increasing the polarization rate and electron density in the structure, active site concentration, and promoting oxygen activation ability. Notwithstanding, there is yet a huge knowledge break in the design, preparation, detection, application, and prospect of cyano-rich g-C3 N4 . Accordingly, an overall review is arranged to substantially comprehend the research progress and position of cyano-rich g-C3 N4 materials. An overall overview of the current research position in the synthesis, characterization (determination of their location and quantity), application, and reaction mechanism analysis of cyano-rich g-C3 N4 materials to provide a quantity of novel suggestions for cyano-modified carbon nitride materials' construction is provided. In view of the prevailing challenges and outlooks of cyano-rich g-C3 N4 materials, this paper will purify the growth direction of cyano-rich g-C3 N4 , to achieve a more in-depth exploration and broaden the applications of cyano-rich g-C3 N4 .

DEAD-BOX RNA HELICASE 27 regulates microRNA biogenesis, zygote division, and stem cell homeostasis.

After double fertilization, zygotic embryogenesis initiates a new life cycle, and stem cell homeostasis in the shoot apical meristem (SAM) and root apical meristem (RAM) allows plants to produce new tissues and organs continuously. Here, we report that mutations in DEAD-BOX RNA HELICASE 27 (RH27) affect zygote division and stem cell homeostasis in Arabidopsis (Arabidopsis thaliana). The strong mutant allele rh27-1 caused a zygote-lethal phenotype, while the weak mutant allele rh27-2 led to minor defects in embryogenesis and severely compromised stem cell homeostasis in the SAM and RAM. RH27 is expressed in embryos from the zygote stage, and in both the SAM and RAM, and RH27 is a nucleus-localized protein. The expression levels of genes related to stem cell homeostasis were elevated in rh27-2 plants, alongside down-regulation of their regulatory microRNAs (miRNAs). Further analyses of rh27-2 plants revealed reduced levels of a large subset of miRNAs and their pri-miRNAs in shoot apices and root tips. In addition, biochemical studies showed that RH27 associates with pri-miRNAs and interacts with miRNA-biogenesis components, including DAWDLE, HYPONASTIC LEAVES 1, and SERRATE. Therefore, we propose that RH27 is a component of the microprocessor complex and is critical for zygote division and stem cell homeostasis.

Mediation of White Matter Alterations in the Association Between Ventricular Dilation and Cognitive Decline in Hydrocephalus Patients: An MRI Study.

BACKGROUND: Cognitive impairment is commonly observed in hydrocephalus patients. Ventricular enlargement compresses brain parenchyma, especially the white matter (WM). PURPOSE: To investigate whether the relationship between ventricular dilation and cognitive decline in hydrocephalus patients is mediated by WM alterations. STUDY TYPE: Retrospective. POPULATION: 51 communicating hydrocephalus patients (median age, 54 years), 50 obstructive hydrocephalus patients (median age, 49 years), and 53 control subjects (median age, 50 years). FIELD STRENGTH/SEQUENCE: Diffusion tensors imaging, 3D T1 BRAVO, 3D FIESTA, CUBE T2, and FLAIR sequences at 3T. ASSESSMENT: DTI parameters (skeletonized fractional anisotropy (FA), skeletonized mean diffusivity (MD), and peak width of skeletonized mean diffusivity p(PSMD)) were extracted using FSL software. Global, periventricular, and deep white matter hyperintensity (WMH) volumes, degree of ventricular enlargement (Evans index), and other conventional imaging markers (number of lacunes and perivascular spaces, intracranial and brain volume) were extracted using united imaging intelligence. Cognitive tests included Montreal cognitive assessment (MoCA), clock drawing test (CDT), and vocabulary fluency test (VFT). STATISTICAL TESTS: Multivariable linear regression analysis, mediation analyses, and dominance analysis. P-value <0.05 was considered significant. RESULTS: The degree of ventricular dilation, DTI parameters, and cognitive function scores were interrelated. The skeletonized FA values (ß = -0.0917, 95% confidence interval (CI): -0.205, -0.024) and normalized global WMH volume (ß = -0.0635, 95% CI: -0.13, -0.0005) together mediated 37.2% of the association between Evans index and MoCA. A comparable causal pathway was found for periventricular WMHs but not for deep WMHs. Dominance analysis indicated skeletonized FA values had a greater impact on cognition than WMH volume. The skeletonized FA values also mediated the association between Evans index and CDT (ß = -0.0897, 95% CI: -0.165, -0.026) and VFT (ß = -0.1589, 95% CI: -0.27, -0.083). CONCLUSION: WM alterations were causal mediators between ventricular dilation and cognitive decline in hydrocephalus patients. EVIDENCE LEVEL: 3. TECHNICAL EFFICACY: Stage 3.

Evaluation of pancreatic iodine uptake and related influential factors in multiphase dual-energy CT.

OBJECTIVES: To establish normative values and identify potential factors influencing pancreatic iodine uptake using dual-energy CT (DECT). MATERIALS AND METHODS: This retrospective study included participants without pancreatic diseases undergoing DECT at two institutions with different platforms. Their protocols both included arterial phase (AP), portal venous phase (PP), and equilibrium phase (EP), defined as 35 s-40 s, 60 s-70 s, and 150 s-180 s after injection of contrast agent, respectively. Both iodine concentration (IC) and normalised IC (NIC) were measured. Demographic features, local measurements of the pancreas and visceral fat area (VFA) were considered as potential factors influencing iodine uptake using multivariate linear regression analyses. RESULTS: A total of 562 participants (median age 58 years [interquartile range: 47-67], with 282 men) were evaluated. The mean IC differed significantly between two institutions (all p < 0.001) across three contrast-enhanced phases, while the mean NIC showed no significant differences (all p > 0.05). The mean values of NIC were 0.22 at AP, 0.43 at PP and 0.45 at EP. NICAP was independently affected by VFA (ß = 0.362, p < 0.001), smoking (ß = -0.240, p = 0.001), and type-II diabetes (ß = -0.449, p < 0.001); NICPP by VFA (ß = -0.301, p = 0.017) and smoking (ß = -0.291, p < 0.001); and NICEP by smoking (ß = -0.154, p = 0.10) and alcohol consumption (ß = -0.350, p < 0.001) with statistical power values over 0.81. CONCLUSION: NIC values were consistent across institutions. Abdominal obesity, smoking, alcohol consumption, and diabetes are independent factors influencing pancreatic iodine uptake. CLINICAL RELEVANCE STATEMENT: This study has provided reference normative values, influential factors and effective normalisation methods of pancreatic iodine uptake in multiphase dual-energy CT for future studies in this area as a new biological marker. KEY POINTS: Evaluation of pancreatic iodine uptake measured by dual-energy CT is a promising method for future studies. Abdominal obesity, smoking, alcohol consumption, diabetes, and sex are independent factors influencing pancreatic iodine uptake. Utility of normalised iodine concentration is necessary to ensure the consistency across different institutions.

MicroRNAs regulate the vicious cycle of vascular calcification-osteoporosis in postmenopausal women.

Menopause is a normal physiological process accompanied by changes in various physiological states. The incidence of vascular calcification (VC) increases each year after menopause and is closely related to osteoporosis (OP). Although many studies have investigated the links between VC and OP, the interaction mechanism of the two under conditions of estrogen loss remains unclear. MicroRNAs (miRNAs), which are involved in epigenetic modification, play a critical role in estrogen-mediated mineralization. In the past several decades, miRNAs have been identified as biomarkers or therapeutic targets in diseases. Thus, we hypothesize that these small molecules can provide new diagnostic and therapeutic approaches. In this review, we summarize the close interactions between VC and OP and the role of miRNAs in their interplay.

Remote ischemic conditioning reduces adverse events in patients with acute ischemic stroke complicating acute myocardial infarction: a randomized controlled trial.

BACKGROUND: Acute ischemic stroke (AIS) complicating an acute myocardial infarction (AMI) is not uncommon, but can severely worsen the clinical prognosis. This study aimed to investigate whether remote ischemic conditioning (RIC) could provide clinical benefits to patients with AIS complicating AMI. METHODS: Subjects with AIS complicating AMI were recruited in this double-blind, randomized, controlled trial; assigned to the RIC and sham groups; and respectively underwent twice daily RIC and sham RIC for 2 weeks. All subjects received standard medical therapy. The primary endpoint was the rate of major adverse cardiac and cerebrovascular events (MACCEs) within 3 months after enrollment. MACCEs comprise of death from all causes, unstable anginas, AMI, acute ischemic strokes, and transient ischemic attacks. RESULTS: Eighty subjects were randomly assigned; 37 patients in the RIC group and 40 patients in the sham-RIC group completed the 3-month follow-up and were included in the final analysis. Both RIC and sham RIC procedures were well tolerated. At 3-month follow-up, 11 subjects (29.7%) in the RIC group experienced MACCEs compared to 21 (52.5%) in the sham group (hazard ratio [HR], 0.396; 95% confidence interval, 0.187-0.838; adjusted p < 0.05). Six subjects (16.2%) in the RIC group had died at the 3-month follow up, significantly lower than the 15 (37.5%) deaths in the sham group (adjusted HR 0.333; 95% CI 0.126-0.881; p = 0.027). Seventeen subjects (45.9%) in the RIC group and 6 subjects (15.0%) in the sham group achieved functional independence (mRS score ≤ 2) at 3-month follow-up (adjusted OR 12.75; 95% CI 2.104-77.21; p = 0.006). CONCLUSIONS: Among patients with acute ischemic stroke complicating acute myocardial infarction, treatment with remote ischemic conditioning decreased the major adverse cardiac and cerebrovascular events and improved functional outcomes at 90 days. TRIAL REGISTRATION: URL: www. CLINICALTRIALS: gov . Unique identifier: NCT03868007. Registered 8 March 2019.

Deep Mind 21 functional does not extrapolate to transition metal chemistry.

The development of density functional approximations stands at a crossroads: while machine-learned functionals show potential to surpass their human-designed counterparts, their extrapolation to unseen chemistry lags behind. Here we assess how well the recent Deep Mind 21 (DM21) machine-learned functional [Science, 2021, 374, 1385-1389], trained on main-group chemistry, extrapolates to transition metal chemistry (TMC). We show that DM21 demonstrates comparable or occasionally superior accuracy to B3LYP for TMC, but consistently struggles with achieving self-consistent field convergence for TMC molecules. We also compare main-group and TMC machine-learning DM21 features to shed light on DM21's challenges in TMC. We finally propose strategies to overcome limitations in the extrapolative capabilities of machine-learned functionals in TMC.

Association analysis of the sorting nexin 29 (SNX29) gene copy number variations with growth traits in Diannan small-ear (DSE) pigs.

SNX29 is a potential functional gene associated with meat production traits. Previous studies have shown that SNX29 copy number variation (CNV) could be implicated with phenotype in goats. However, in Diannan small-ear (DSE) pigs, the genetic impact of SNX29 CNV on growth traits remains unclear. Therefore, this study investigated the associations between SNX29 CNVs (CNV10810 and CNV10811) and growth traits in 415 DSE pigs. The results revealed that the CNV10810 mutation was significantly associated with backfat thickness in DSE pigs at 12 and 15 months old (P < 0.05), while the CNV10811 mutation had significant effects on various growth traits at 6 and 12 months old, particularly for body weight, body height, back height and backfat thickness (P < 0.05 or P < 0.001). In conclusion, our results confirm that SNX29 CNV plays a role in regulating growth and development in pigs, thus suggesting its potential application for pig breeding programmes.

Distributed Vibration Monitoring System for 10 kV-400 kVA 3D Wound Core Transformer under Progressive Short-Circuit Impulses.

As large-scale, high-proportion, and efficient distribution transformers surge into the grids, anti-short circuit capability testing of transformer windings in efficient distribution seems necessary and prominent. To deeply explore the influence of progressively short-circuit shock impulses on the core winding deformation of efficient power transformers, a finite element theoretical model was built by referring to a three-phase three-winding 3D wound core transformer with a model of S20-MRL-400/10-NX2. The distributions of internal equivalent force and total deformation of the 3D wound core transformer along different paths under progressively short-circuit shock impulses varying from 60% to 120% were investigated. Results show that the equivalent stress and total deformation change rate reach their maximum as the short-circuit current increases from 60% to 80%, and the maximum and average variation rate for the equivalent stress reach 177.75% and 177.43%, while the maximum and average variation rate for the total deformation corresponds to 178.30% and 177.45%, respectively. Meanwhile, the maximum equivalent stress and maximum total deformation reach 29.81 MPa and 38.70 µm, respectively, as the applied short-circuit current increased to 120%. In light of the above observations, the optimization and deployment of wireless sensor nodes was suggested. Therefore, a distributed monitoring system was developed for acquiring the vibration status of the windings in a 3D wound core transformer, which is a beneficial supplement to the traditional short-circuit reactance detection methods for an efficient grid access spot-check of distribution transformers.

An Improved Nested U-Net Network for Fluorescence In Situ Hybridization Cell Image Segmentation.

Fluorescence in situ hybridization (FISH) is a powerful cytogenetic method used to precisely detect and localize nucleic acid sequences. This technique is proving to be an invaluable tool in medical diagnostics and has made significant contributions to biology and the life sciences. However, the number of cells is large and the nucleic acid sequences are disorganized in the FISH images taken using the microscope. Processing and analyzing images is a time-consuming and laborious task for researchers, as it can easily tire the human eyes and lead to errors in judgment. In recent years, deep learning has made significant progress in the field of medical imaging, especially the successful application of introducing the attention mechanism. The attention mechanism, as a key component of deep learning, improves the understanding and interpretation of medical images by giving different weights to different regions of the image, enabling the model to focus more on important features. To address the challenges in FISH image analysis, we combined medical imaging with deep learning to develop the SEAM-Unet++ automated cell contour segmentation algorithm with integrated attention mechanism. The significant advantage of this algorithm is that it improves the accuracy of cell contours in FISH images. Experiments have demonstrated that by introducing the attention mechanism, our method is able to segment cells that are adherent to each other more efficiently.

The Dynamic Evolution Model of the Chemical and Carbon Isotopic Composition of C1-3 during the Hydrocarbon Generation Process.

A new approach is presented in this paper for the dynamic modeling of the chemical and isotopic evolution of C1-3 during the hydrocarbon generation process. Based on systematic data obtained from published papers for the pyrolysis of various hydrocarbon sources (type I kerogen/source rock, type II kerogen/source rock, type III kerogen/source rock, crude oil, and asphalt, etc.), the empirical evolution framework of the chemical and isotopic composition of C1-3 during the hydrocarbon generation process was built. Although the empirical framework was built only by fitting a large amount of pyrolysis data, the chemical and isotopic composition of C1-3 derived from the pyrolysis experiments all follow evolution laws, convincing us that it is applicable to the thermal evolution process of various hydrocarbon sources. Based on the simplified formula of the isotopic composition of mixed natural gas at different maturities (δ13Cmixed), δ13Cmixed = X×niA×δ13CiA+Y×niB×δ13CiBX×niA+Y×niB, it can be derived that the cumulative isotopic composition of alkane generated in a certain maturity interval can be expressed by the integral of the product of the instantaneous isotopic composition and instantaneous yield at a certain maturity point, and then divided by the cumulative yield of alkane generated in the corresponding maturity interval. Thus, the cumulative isotopic composition (A(X)), cumulative yield (B(X)), instantaneous isotope (C(X)), and instantaneous yield (D(x)) in the dynamic model, comply with the following formula during the maturity interval of (X0~X). A(X) = ∫X0XCX×DXdxB(X), where A(X) and B(X) can be obtained by the fitting of pyrolysis data, and D(x) can also be obtained from the derivation of B(X). The dynamic model was applied on the pyrolysis data of Pingliang Shale to illustrate the quantitative evolution of the cumulative yield, instantaneous yield, cumulative isotope, and instantaneous isotope of C1-3 with increasing maturity. The dynamic model can quantify the yield of methane, ethane, and propane, as well as δ13C1, δ13C2, and δ13C3, respectively, during the hydrocarbon generation process. This model is of great significance for evaluating the natural gas resources of hydrocarbon source rock of different maturities and for identifying the origin and evolutionary process of hydrocarbons by chemical and isotopic data. Moreover, this model provides an approach to study the dynamic evolution of the isotope series of C1-3 (including reversed isotopic series), which is promising for revealing the mechanism responsible for isotopic reversal when combined with post-generation studies.

Redefining the Scope of Targeted Protein Degradation: Translational Opportunities in Hijacking the Autophagy-Lysosome Pathway.

The advent of multi-specific targeted protein degradation (TPD) therapies has made it possible to drug targets that have long been considered to be inaccessible. For this reason, the foremost TPD modalities - molecular glues and proteolysis targeting chimeras (PROTACs) -have been widely adopted and developed in therapeutic programs across the pharmaceutical and biotechnology industries. While there are many clear advantages to these two approaches, there are also blind spots. Specifically, PROTACs and molecular glues are inherently mechanistically analogous in that targets of both are degraded via the 26s proteasome; however, not all disease-relevant targets are suitable for ubiquitin proteasome system (UPS)-mediated degradation. The alternative mammalian protein degradation pathway, the autophagy-lysosome system (or ALS), is capable of degrading targets that elude the UPS such as long-lived proteins, insoluble protein aggregates, and even abnormal organelles. Emerging TPD strategies- such as ATTEC, AUTAC, and LYTAC- take advantage of the substrate diversity of the ALS to greatly expand the clinical utility of TPD. In this Perspective, we will discuss the array of current TPD modalities, with a focus on critical evaluation of these novel ALS-mediated degradation techniques.

Ultraviolet Photodissociation Reveals the Molecular Mechanism of Crown Ether Microsolvation Effect on the Gas-Phase Native-like Protein Structure.

Maintaining the protein high-order structures and interactions during the transition from aqueous solution to gas phase is essential to the structural analysis of native mass spectrometry (nMS). Herein, we systematically interrogate the effects of charge state and crown ether (CE) complexation on the gas-phase native-like protein structure by integrating nMS with 193 nm ultraviolet photodissociation (UVPD). The alterations of photofragmentation yields of protein residues and the charge site distribution of fragment ions reveal the specific sites and sequence regions where charge and CE take effect. Our results exhibit the CE complexation on protonated residues can largely alleviate the structure disruption induced by the intramolecular solvation of charged side chains. The influences of CE complexation and positive charge on gas-phase protein structure exhibit generally opposite trends because the CE microsolvation avoids the hydrogen-bonding formation between the charged side chains with backbone carbonyls. Thus, CE complexation leads to a more stable and native-like protein structure in the gas phase.

Structural Mass Spectrometry Probes the Inhibitor-Induced Allosteric Activation of CDK12/CDK13-Cyclin K Dissociation.

The rational design and development of effective inhibitors for cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) are largely dependent on the understanding of the dynamic inhibition conformations but are difficult to be achieved by conventional characterization tools. Herein, we integrate the structural mass spectrometry (MS) methods of lysine reactivity profiling (LRP) and native MS (nMS) to systematically interrogate both the dynamic molecular interactions and overall protein assembly of CDK12/CDK13-cyclin K (CycK) complexes under the modulation of small molecule inhibitors. The essential structure insights, including inhibitor binding pocket, binding strength, interfacial molecular details, and dynamic conformation changes, can be derived from the complementary results of LRP and nMS. We find the inhibitor SR-4835 binding can greatly destabilize the CDK12/CDK13-CycK interactions in an unusual allosteric activation way, thereby providing a novel alternative for the kinase activity inhibition. Our results underscore the great potential of LRP combination with nMS for the evaluation and rational design of effective kinase inhibitors at the molecular level.