Structure and function of the apical PIKKs in double-strand break repair.

Members of the phosphatidylinositol 3' kinase (PI3K)-related kinases (PIKKs) family, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), ataxia-telangiectasia mutated and Rad3-related (ATR), mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein 1 (TRRAP/Tra1), participate in a variety of physiological processes, such as cell-cycle control, metabolism, transcription, replication, and the DNA damage response. In eukaryotic cells, DNA-PKcs, ATM, and ATR-ATRIP are the main sensors and regulators of DNA double-strand break repair. The purpose of this review is to describe recent structures of DNA-PKcs, ATM, and ATR, as well as their functions in activation and phosphorylation in different DNA repair pathways.

Impact of perception reduction of employment opportunities on employment pressure of college students under COVID-19 epidemic-joint moderating effects of employment policy support and job-searching self-efficacy.

Based on the stress interaction theory, this research constructed a model to study the joint moderating effects of the perception reduction of employment opportunities under the COVID-19 epidemic on the employment pressure of college students. With two moderating variables introduced, employment policy support and job-searching self-efficacy, this research studied the mechanism and boundary conditions of perception reduction of employment opportunities on employment pressure of college students from both individual and environmental aspects. The study found that during the epidemic if college students perceived fewer employment opportunities, they could have greater employment pressure from themselves, schools, and families; and that under the joint moderation of employment policy support and job-searching self-efficacy, the perception reduction of employment opportunities under the COVID-19 epidemic, the employment pressure of college students, universities, and families were connected, with different adjustment mechanisms. Based on empirical data, this research can provide theoretical enlightenment and practical guidance for the government, universities, and families to alleviate the employment pressure on college students during the epidemic.

Anatomical and Imaging Study on the Optimum Entry Point and Trajectory for Anterior Transpedicular Root Screw Placement into the Lower Cervical Spine.

Objective: To study the optimum entry point and trajectory for anterior transpedicular root screw (ATPRS) placement into the lower cervical spine (LCS), so as to provide a basis for clinical application. Methods: A retrospective analysis of cervical CT images of patients who underwent cervical CT examination in the Spinal Surgery of Ningbo No. 6 Hospital from January 2020 to August 2021 was conducted. The data were obtained and modeled. On the coronal plane, the vertebral body (VB) between the anterior midline of cervical vertebral segments C3-7 and the left P line (by drawing the line parallel to the anterior midline of the VB at the intersection of the anterior edge of the Luschka's joint and the upper endplate) was equally divided into 9 zones (a-i). The ideal entry point and path of cervical ATPRS were designed and recorded. Additionally, 7 cadaveric specimens were selected, and the screw placement parameters were regenerated according to the above methods for screw placement. Results: Zone i of each segment, with the longest screw length, was the best area for screw placement. In all patients, the horizontal angles of vertebrae C3-7 in zones a, d, and g, zones b, e, and h, and zones c, f, and i showed a gradually decreasing trend. The sagittal angle range of C3-7 in all patients showed a gradually increasing trend in zones a-c, d-f, and g-i. The distance from the anterior midline of C3-7 to the P line increased in all patients, and the distance was longer in males than in females, with statistical significance. Pedicle screws were successfully inserted in all the 7 cadaveric specimens. Conclusions: ATPRS placement can be used for LCS internal fixation, and the precise screw placement parameters can be simulated by the software, which provides theoretical basis for its future clinical application.

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination.

Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51-DNA and Dmc1-DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.

Age-related increase in muscle stiffness is muscle length dependent and associated with muscle force in senior females.

BACKGROUND: In aging, muscle stiffness is considered as one of the factors associated with the reduction of force generation capability. There have been inconsistent findings on age-related alteration in the passive stiffness of quadriceps muscle in the female adults. Thus, the aim of this study was to determine the effect of aging on the shear moduli of the superficial muscle heads of the quadriceps and to explore its relationship with knee extension force. METHODS: Passive shear moduli of the rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) were measured at rest using shear wave elastography in 20 young and 20 senior female adults. Measurements were repeated at four knee joint positions, that is, 30°, 60°, 90°, and 105° of knee flexion. Maximal isometric voluntary knee extension force was assessed at 30°, 60°, and 90° of knee flexion. RESULTS: As per our findings, senior adults were determined to have significantly higher passive muscle shear moduli in the RF (by 34% - 68%; all p < 0.05) and the VL muscle heads (by 13%-16%, all p < 0.05) at and beyond 60° of knee flexion. Age-related increase in the VM was evident at 105° knee flexion (by11%, p = 0.020). The RF shear modulus was negatively correlated to the maximal isometric voluntary contraction force measured at 60° (r = - 0.485, p = 0.030) in senior adults. CONCLUSIONS: Senior female adults had greater passive stiffness at the superficial muscle heads of the quadriceps muscles when measured at long muscle length. Among the senior female adults, the passive stiffness of RF has been determined to have a negative association with the knee extensor force only at 60° knee flexion. No significant association was noted for other angles and muscles.

Baicalin suppress growth and virulence-related factors of methicillin-resistant Staphylococcus aureus in vitro and vivo.

A Staphylococcus aureus (S.aureus) was isolated from pigs suffered in pneumonia that can't be cured by antibiotic such as methicillin and vancomycin. It was demonstrated that baicalin, an active natural compound extracted from the traditional Chinese medicinal, possess antimicrobial activity. In the present study, we evaluate it efficacy in vitro and vivo against this isolated methicillin-resistant S.aureus (MRSA). Our findings demonstrated that baicalin can inhibit S. aureus growth in a dose-dependent manner and attenuate the biofilm formation. Scanning electron microscopies showed that cell membrane was damaged and accompany with contents leaks after treated with high concentration of baicalin. In addition, baicalin exerted inhibitory effects on the expression of S.aureus virulence-related factors. Moreover, baicalin treated mice had enhanced survival after a lethal dose of S.aureus infection compared with untreated mice. Simultaneously, the pathological tissue damage and bacterium burden were decrease in baicalin treated mice. These data demonstrated that baicalin displayed a high effectiveness in vitro and vivo against MRSA infection, suggesting that baicalin may potentially be used to treat MRSA infection.

Influenza A virus infection induces liver injury in mice.

Respiratory infections such as SARS-CoV in humans are often accompanied by mild and self-limiting hepatitis. As a respiratory disease, influenza A virus (IAV) infection can lead to hepatitis, but the mechanism remains unclear. This study aimed to investigate the occurrence of hepatitis by establishing a model for infected mice for three different subtypes of respiratory IAVs (H1N1, H5N1, and H7N2). Histological analysis was performed, and results showed increase serum aminotransferase (ALT and AST) levels and evident liver injury on days 3 and 7, especially on day 5 post infection. Immunohistochemistry (IHC) results indicated a wide distribution of IAV's positive signals in the liver of infected mice. Real-time PCR results further revealed a similar viral titer to IHC that presented a remarkedly positive correlation with histology injury. All these data showed that the mouse model suitably contributed valuable information about the mechanism underlying the occurrence of hepatitis induced by respiratory influenza virus.

Relationship between pre-exercise muscle stiffness and muscle damage induced by eccentric exercise.

This study aimed to determine whether pre-exercise muscle stiffness is related to the amount of muscle damage induced by an eccentric exercise and to determine whether the post-exercise increase in stiffness is homogenously distributed between the synergist muscles. Fifty healthy participants were randomly assigned to an eccentric exercise group or a control group. The shear modulus (an index of stiffness) of rectus femoris (RF), vastus lateralis (VL) and vastus medialis oblique (VMO) was measured before, immediately after and at 48 h after eccentric exercise. The maximal isometric voluntary knee extension (MVC) torque was also measured. Significant reduction in MVC torque was observed in the eccentric group both at post and 48 H when compared with pre-exercise (both p < .001). RF shear modulus increased significantly when assessed at 90° of knee flexion at post and 48 H after the eccentric exercise (p = .004 and .005, respectively). Slight but significant decrease in VL shear modulus was observed at post-exercise for the eccentric group (p = .002). No change was observed in VMO. The decrease in MVC at 48 H was negatively correlated with the RF shear modulus measured at 90° of knee flexion before the exercise. Eccentric exercise induced a wide range of peak torque reduction and muscle-head specific modulation on muscle stiffness. Participants with stiffer RF muscles exhibited greater decrease in force generating capacity at 48 H after eccentric exercise.

Stiffness of individual quadriceps muscle assessed using ultrasound shear wave elastography during passive stretching.

BACKGROUND: Until recently it has not been possible to isolate the mechanical behavior of individual muscles during passive stretching. Muscle shear modulus (an index of muscle stiffness) measured using ultrasound shear wave elastography can be used to estimate changes in stiffness of an individual muscle. The aims of the present study were (1) to determine the shear modulus-knee angle relationship and the slack angle of the vastus medialis oblique (VMO), rectus femoris (RF), and vastus lateralis (VL) muscles; (2) to determine whether this differs between the muscles. METHODS: Nine male rowers took part in the study. The shear modulus of VMO, RF, and VL muscles was measured while the quadriceps was passively stretched at 3°/s. The relationship between the muscle shear modulus and knee angle was plotted as shear modulus-knee angle curve through which the slack angle of each muscle was determined. RESULTS: The shear modulus of RF was higher than that of VMO and VL when the muscles were stretched over 54° (all p < 0.01). No significant difference was found between the VMO and VL (all p > 0.05). The slack angle was similar among the muscles: 41.3° ± 10.6°, 44.3° ± 9.1°, and 44.3° ± 5.6° of knee flexion for VMO, RF, and VL, respectively (p = 0.626). CONCLUSION: This is the first study to experimentally determine the muscle mechanical behavior of individual heads of the quadriceps during passive stretching. Different pattern of passive tension was observed between mono- and bi-articular muscles. Further research is needed to determine whether changes in muscle stiffness are muscle-specific in pathological conditions or after interventions such as stretching protocols.

Determining the RAD51-DNA Nucleoprotein Filament Structure and Function by Cryo-Electron Microscopy.

Homologous recombination is a universal tool for DNA double-strand break and replication fork repair, and it is catalyzed by a highly conserved family of recombinases. In eukaryotes, Rad51 is the recombinase that catalyzes the pairing of homologous DNA molecules and the exchange of strands between the paired molecules. Rad51 assembles on single-stranded DNA (ssDNA) stemming from lesion processing to form a right-handed helical polymer that engages then samples double-stranded DNA (dsDNA) for homology. Upon matching with a homologous sequence, the Rad51-bound ssDNA invades the dsDNA, leading to the formation of a DNA joint with concomitant displacement of the strand of like polarity. The Rad51-DNA filaments are amenable to structural studies using cryo-electron microscopy (cryo-EM). In particular, recent technical breakthroughs in cryo-EM have made it possible to define the structure and function of human RAD51 at near-atomic resolution. In this chapter, we describe our cryo-EM approach to capture the human RAD51 filament structures in various stages of catalysis. The approach may also be useful for related recombinases and other helical assemblies.

Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange.

The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) and generates a helical filament that is able to search for and invade homologous double-stranded DNA (dsDNA), thus leading to strand separation and formation of new base pairs between the initiating ssDNA and the complementary strand within the duplex. Here, we used cryo-EM to solve the structures of human RAD51 in complex with DNA molecules, in presynaptic and postsynaptic states, at near-atomic resolution. Our structures reveal both conserved and distinct structural features of the human RAD51-DNA complexes compared with their prokaryotic counterpart. Notably, we also captured the structure of an arrested synaptic complex. Our results provide new insight into the molecular mechanisms of the DNA homology search and strand-exchange processes.