Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Antifouling Asymmetric Block Copolymer Nanofilms via Freestanding Interfacial Polymerization for Efficient and Sustainable Water Purification.

Membrane materials that resist nonspecific or specific adsorption are urgently required in widespread applications. In water purification, inevitable membrane fouling not only limits separation performance, but also remarkably increases operation requirements, and augments extra maintenance costs and higher energy consumption. In this work, we report a freestanding interfacial polymerization (IP) fabrication strategy for in-situ creation of asymmetric block copolymer (BCP) nanofilms with antifouling properties, greatly outperforming the conventional surface post-modification approaches. The resultant asymmetric BCP nanofilms with highly-dense, highly-hydrophilic polyethylene glycol (PEG) brushes, can be readily formed via a typical IP process of a double-hydrophilic BCP composed of an antifouling PEG block and a membrane-forming multiamine block. The asymmetric BCP nanofilms have been applied for efficient and sustainable natural water purification, demonstrating extraordinary antifouling capabilities accompanied with superior separation performance far beyond commercial polyamide nanofiltration membranes. The antifouling behaviors of BCP nanofilms derived from the combined effect of the hydration layer, electrostatic repulsion and steric hindrance were further elucidated by water flux and fouling resistance in combination with all-atom molecular dynamics simulation. This work opens up a new avenue for large-scale and low-cost creation of broad-spectrum, asymmetric membrane materials with diverse functional "defect-free" surfaces in real-world applications.

Loading dynamics of one SARS-CoV-2-derived peptide into MHC-II revealed by kinetic models.

Major histocompatibility complex class II (MHC-II) plays an indispensable role in activating CD4+ T cell immune responses by presenting antigenic peptides on the cell surface for recognition by T cell receptors. The assembly of MHC-II and antigenic peptide is therefore a prerequisite for the antigen presentation. To date, however, the atomic-level mechanism underlying the peptide-loading dynamics for MHC-II is still elusive. Here, by constructing Markov state models based on extensive all-atom molecular dynamics simulations, we reveal the complete peptide-loading dynamics into MHC-II for one SARS-CoV-2 S-protein-derived antigenic peptide (235ITRFQTLLALHRSYL249). Our Markov state model identifies six metastable states (S1-S6) during the peptide-loading process and determines two dominant loading pathways. The peptide could potentially approach the antigen-binding groove via either its N- or C-terminus. Then, the consecutive insertion of several anchor residues into the binding pockets profoundly dictates the peptide-loading dynamics. Notably, the MHC-II αA52-E55 motif could guide the peptide loading into the antigen-binding groove via forming ß-sheets conformation with the incoming peptide. The rate-limiting step, namely S5→S6, is mainly attributed to a considerable desolvation penalty triggered by the binding of the peptide C-terminus. Moreover, we further examined the conformational changes associated with the peptide exchange process catalyzed by the chaperon protein HLA-DM. A flipped-out conformation of MHC-II αW43 captured in S1-S3 is considered a critical anchor point for HLA-DM to modulate the structural dynamics. Our work provides deep structural insights into the key regulatory factors in MHC-II responsible for peptide recognition and guides future design for peptide vaccines against SARS-CoV-2.

Phosphorylation Regulation Mechanism of ß2 Integrin for the Binding of Filamin Revealed by Markov State Model.

Leukocyte adhesion deficiency-1 (LAD-1) disorder is a severe immunodeficiency syndrome caused by deficiency or mutation of ß2 integrin. The phosphorylation on threonine 758 of ß2 integrin acts as a molecular switch inhibiting the binding of filamin. However, the switch mechanism of site-specific phosphorylation at the atom level is still poorly understood. To resolve the regulation mechanism, all-atom molecular dynamics simulation and Markov state model were used to study the dynamic regulation pathway of phosphorylation. Wild type system possessed lower binding free energy and fewer number of states than the phosphorylated system. Both systems underwent local disorder-to-order conformation conversion when achieving steady states. To reach steady states, wild type adopted less number of transition paths/shortest path according to the transition path theory than the phosphorylated system. The underlying phosphorylated regulation pathway was from P1 to P0 and then P4 state, and the main driving force should be hydrogen bond and hydrophobic interaction disturbing the secondary structure of phosphorylated states. These studies will shed light on the pathogenesis of LAD-1 disease and lay a foundation for drug development.

Dynamics of peptide loading into major histocompatibility complex class I molecules chaperoned by TAPBPR.

Major histocompatibility complex class I (MHC-I) molecules display antigenic peptides on the cell surface for T cell receptor scanning, thereby activating the immune response. Peptide loading into MHC-I molecules is thus a critical step during the antigen presentation process. Chaperone TAP-binding protein related (TAPBPR) plays a critical role in promoting high-affinity peptide loading into MHC-I, by discriminating against the low-affinity ones. However, the complete peptide loading dynamics into TAPBPR-bound MHC-I is still elusive. Here, we constructed kinetic network models based on hundreds of short-time MD simulations with an aggregated simulation time of ∼21.7 µs, and revealed, at atomic level, four key intermediate states of one antigenic peptide derived from melanoma-associated MART-1/Melan-A protein during its loading process into TAPBPR-bound MHC-I. We find that the TAPBPR binding at the MHC-I pocket-F can substantially reshape the distant pocket-B via allosteric regulations, which in turn promotes the following peptide N-terminal loading. Intriguingly, the partially loaded peptide could profoundly weaken the TAPBPR-MHC stability, promoting the dissociation of the TAPBPR scoop-loop (SL) region from the pocket-F to a more solvent-exposed conformation. Structural inspections further indicate that the peptide loading could remotely affect the SL binding site through both allosteric perturbations and direct contacts. In addition, another structural motif of TAPBPR, the jack hairpin region, was also found to participate in mediating the peptide editing. Our study sheds light on the detailed molecular mechanisms underlying the peptide loading process into TAPBPR-bound MHC-I and pinpoints the key structural factors responsible for dictating the peptide-loading dynamics.

Computational investigations on target-site searching and recognition mechanisms by thymine DNA glycosylase during DNA repair process.

DNA glycosylase, as one member of DNA repair machineries, plays an essential role in correcting mismatched/damaged DNA nucleotides by cleaving the N-glycosidic bond between the sugar and target nucleobase through the base excision repair (BER) pathways. Efficient corrections of these DNA lesions are critical for maintaining genome integrity and preventing premature aging and cancers. The target-site searching/recognition mechanisms and the subsequent conformational dynamics of DNA glycosylase, however, remain challenging to be characterized using experimental techniques. In this review, we summarize our recent studies of sequential structural changes of thymine DNA glycosylase (TDG) during the DNA repair process, achieved mostly by molecular dynamics (MD) simulations. Computational simulations allow us to reveal atomic-level structural dynamics of TDG as it approaches the target-site, and pinpoint the key structural elements responsible for regulating the translocation of TDG along DNA. Subsequently, upon locating the lesions, TDG adopts a base-flipping mechanism to extrude the mispaired nucleobase into the enzyme active-site. The constructed kinetic network model elucidates six metastable states during the base-extrusion process and suggests an active role of TDG in flipping the intrahelical nucleobase. Finally, the molecular mechanism of product release dynamics after catalysis is also summarized. Taken together, we highlight to what extent the computational simulations advance our knowledge and understanding of the molecular mechanism underlying the conformational dynamics of TDG, as well as the limitations of current theoretical work.

Structural and mechanistic investigations on CC bond forming α-oxoamine synthase allowing L-glutamate as substrate.

Carbon­carbon (C-C) bonds serve as the fundamental structural backbone of organic molecules. As a critical CC bond forming enzyme, α-oxoamine synthase is responsible for the synthesis of α-amino ketones by performing the condensation reaction between amino acids and acyl-CoAs. We previously identified an α-oxoamine synthase (AOS), named as Alb29, involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072. This enzyme belongs to the α-oxoamine synthase family, a subfamily under the pyridoxal 5'-phosphate (PLP) dependent enzyme superfamily. In this study, we report the crystal structures of Alb29 bound to PLP and L-Glu, which provide the atomic-level structural insights into the substrate recognition by Alb29. We discover that Alb29 can catalyze the amino transformation from L-Gln to L-Glu, besides the condensation of L-Glu with ß-methylcrotonyl coenzyme A. Subsequent structural analysis has revealed that one flexible loop in Alb29 plays an important role in both amino transformation and condensation. Based on the crystal structure of the S87G mutant in the loop region, we capture two distinct conformations of the flexible loop in the active site, compared with the wild-type Alb29. Our study offers valuable insights into the catalytic mechanism underlying substrate recognition of Alb29.

Legumain inhibitor prevents breast cancer bone metastasis by attenuating osteoclast differentiation and function.

Breast cancer is the main lethal disease among females, and metastasis to lung and bone poses a serious threat to patients' life. Therefore, identification of novel molecular mediators that can potentially be exploited as therapeutic targets for treating osteolytic bone metastases is needed. A murine model of breast cancer bone metastasis was developed by injection of 4 T1.2 cells into the left ventricle and hence directly into the arterial system leading to bone. AEP (Asparagine endopeptidase) inhibitor combined with epirubicin or epirubicin alone was administered by intraperitoneal injection into animal model. The presence of bone metastatic and osteolytic lesions in bone were assessed by bioluminescent imaging and X-rays analysis. The expression of EMT (Epithelial-Mesenchymal Transition) relevant genes were examined by Western blotting. Cell migration and invasion were investigated with a transwell assay. Compound BIC-113, small molecule inhibitors of AEP, inhibited AEP enzymatic activity in breast cancer cell lines, and affected invasion and migration of cancer cells, but had no effect on cell growth. In animal model of breast cancer bone metastasis, compound BIC-113 combined with epirubicin inhibited breast cancer bone metastasis and attenuated breast cancer osteolytic lesions in bone by inhibiting osteoclast differentiation and EMT. These results indicate that compound BIC-113 combined with epirubicin has the potential to be used in breast cancer therapy by preventing bone metastasis via improving E-cadherin expression and inhibition of osteoclast formation.

Early aggregation mechanism of Aß16-22 revealed by Markov state models.

Aß16-22 is believed to have critical role in early aggregation of full length amyloids that are associated with the Alzheimer's disease and can aggregate to form amyloid fibrils. However, the early aggregation mechanism is still unsolved. Here, multiple long-term molecular dynamics simulations combining with Markov state model were used to probe the early oligomerization mechanism of Aß16-22 peptides. The identified dimeric form adopted either globular random-coil or extended ß-strand like conformations. The observed dimers of these variants shared many overall conformational characteristics but differed in several aspects at detailed level. In all cases, the most common type of secondary structure was intermolecular antiparallel ß-sheets. The inter-state transitions were very frequent ranges from few to hundred nanoseconds. More strikingly, those states which contain fraction of ß secondary structure and significant amount of extended coiled structures, therefore exposed to the solvent, were majorly participated in aggregation. The assembly of low-energy dimers, in which the peptides form antiparallel ß sheets, occurred by multiple pathways with the formation of an obligatory intermediates. We proposed that these states might facilitate the Aß16-22 aggregation through a significant component of the conformational selection mechanism, because they might increase the aggregates population by promoting the inter-chain hydrophobic and the hydrogen bond contacts. The formation of early stage antiparallel ß sheet structures is critical for oligomerization, and at the same time provided a flat geometry to seed the ordered ß-strand packing of the fibrils. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aß aggregation and will insight into early oligomerization for large ß amyloids.

[The effects of electrostimulation upon soldier's circadian of melatonin in plasma].

Physiological and behavioral rhythms are governed by an endogenous circadian clock. In this paper are reported the studies on soldier's circadian of melatonin concentrations by means of electricity and light pulses presented to the popliteal region (behind the knee). The results showed that the phase of melatonin concentrations can be regulated by both the electricity pulses and the light pulses. A systematic relation was found between the timing of the electro-stimulating and the magnitude and direction of phase shifts, resulting in the generation of a phase response curve. The phase response curve displayed photic model. These findings have implications for the development of more effective treatments for sleep and circadian rhythm disorders.

[Experimental study of electroacupuncture improving the obstruction of vestibular microcirculation in vertebrobasilar insufficiency and the effect on vestibulo-ocular reflex].

AIM: To investigate the mechanism of EA improving the obstruction of inner ear microcirculation and the effect on the vestibulo-ocular reflex (VOR) by comparing the effects of electroacupuncture (EA) with sibelium (flunarizine hydrochloride) on vertebrobasilar insufficiency(VBI). METHODS: Injected with sclerosant-775 injection into the solt tissue on the left side of cervical vertebral transverse processes of rabbits to set up the vertebral artery type of cervical spondylosis (VCS) models. Electronystagmography (ENG) induced by linear acceleration (LA) and horizontal rotation (HR), the transcranial Doppler (TCD), laser Doppler flowmetry (LDF) and hemorheology were used to measure changes of the frequencies of nystagmus, the hemodynamics in the basilar artery (BA), inner ear blood flow(IEBF) and blood viscosity in VBI rabbits. RESULTS: The frequencies of ENG, the velocity of blood flow in BA and IEBF decreased obviously, and whole blood middle where viscosity, whole blood lower where viscosity and erythrocyte distortion index ( EDI) increased significantly in the model group. Sibelium could reduce whole blood viscosity and EDI, and increased the systolic phase velocity (Vs) of blood flow in BA, but had no effect on diastolic phase velocity (Vd) and mean velocity (Vm). EA could not reduce the viscosity of blood and EDI, but had more significant effects on improving IEBF and ENG induced by LA than those of sibelium,and had the tendency of increasing Vs, Vd and Vm. EA and sibelium had no effect on improving ENG induced by HR. CONCLUSION: Inner ear microcirculation obstruction caused by VBI can induce dysfunctions of vestibule cyst macula and horizontal semicircular canals. EA may depend upon the neurohumoral regulation to improve VBI, and ameliorate inner ear blood supply obstruction by enhancing mechanism of local adjusting for microcirculation in the inner ear to recover vestibular cyst macula irritability for LA chiefly. There exist complicated mechanism that EA adjusts blood flow distribution and vestibular signal transduction in vestibular organ in VBI model likely, and remain to be researched deeply.

[Effects of acupuncture at different periods on circadian rhythms of locomotor activity and core body temperature in hamsters].

OBJECTIVE: To explore the time law of electroacupuncture in regulation of circadian rhythms of the organism. METHODS: Effects of electroacupuncture at "Shenshu" (BL 23) at Zi, Wu, Mao and You periods on circadian rhythms of locomotor activity and core body temperature in hamsters were observed with chronobiological research methods. RESULTS: Electroacupuncture at Wu period could decrease the amplitude of locomotor activity rhythm (P < 0.05), at Mao period could delay the peak phase of circadian rhythm and at You period could advance the peak phase of circadian rhythm (both P < 0.05); and electroacupuncture at Mao period could delay 22.36 degrees and at You period advance 39.32 degrees for the rhythm peak of the circadian rhythm of core body temperature. CONCLUSION: Acupuncture has a certain effect on circadian rhythm of locomotor activity and core body temperature.