The clock gene Per1 may exert diurnal control over hippocampal memory consolidation.

The circadian system influences many different biological processes, including memory performance. While the suprachiasmatic nucleus (SCN) functions as the brain's central pacemaker, downstream "satellite clocks" may also regulate local functions based on the time of day. Within the dorsal hippocampus (DH), for example, local molecular oscillations may contribute to time-of-day effects on memory. Here, we used the hippocampus-dependent Object Location Memory task to determine how memory is regulated across the day/night cycle in mice. First, we systematically determined which phase of memory (acquisition, consolidation, or retrieval) is modulated across the 24 h day. We found that mice show better long-term memory performance during the day than at night, an effect that was specifically attributed to diurnal changes in memory consolidation, as neither memory acquisition nor memory retrieval fluctuated across the day/night cycle. Using RNA-sequencing we identified the circadian clock gene Period1 (Per1) as a key mechanism capable of supporting this diurnal fluctuation in memory consolidation, as learning-induced Per1 oscillates in tandem with memory performance in the hippocampus. We then show that local knockdown of Per1 within the DH impairs spatial memory without affecting either the circadian rhythm or sleep behavior. Thus, Per1 may independently function within the DH to regulate memory in addition to its known role in regulating the circadian system within the SCN. Per1 may therefore exert local diurnal control over memory consolidation within the DH.

The clock gene Per1 is necessary in the retrosplenial cortex-but not in the suprachiasmatic nucleus-for incidental learning in young and aging male mice.

Aging impairs both circadian rhythms and memory, though the relationship between these impairments is not fully understood. Circadian rhythms are largely dictated by clock genes within the body's central pacemaker, the suprachiasmatic nucleus (SCN), though these genes are also expressed in local clocks throughout the body. As circadian rhythms can directly affect memory performance, one possibility is that memory deficits observed with age are downstream of global circadian rhythm disruptions stemming from the SCN. Here, we demonstrate that expression of clock gene Period1 within a memory-relevant cortical structure, the retrosplenial cortex (RSC), is necessary for incidental learning, and that age-related disruption of Period1 within the RSC-but not necessarily the SCN-contributes to cognitive decline. These data expand the known functions of clock genes beyond maintaining circadian rhythms and suggests that age-associated changes in clock gene expression modulates circadian rhythms and memory performance in a brain region-dependent manner.

Micro-elimination of hepatitis C virus infection in the rural and remote areas of Taiwan - A multi-center collaborative care model.

INTRODUCTION: Taiwan has several hepatitis C virus (HCV) hyper-endemic areas. We aimed to evaluate the effectiveness and safety of a collaborative HCV care system with an outreach decentralized strategy among the resource-constrained rural/remote areas of Taiwan. METHODS: The pilot study was conducted in four high HCV-endemic townships in the rural/remote areas of Taoyuan, Alishan, Zhuoxi and Xiulin. Registered residents who worked or lived in the four areas and were aged 30-75 years were invited to participate in this program. Multidisciplinary HCV care teams provided outreach decentralized services of anti-HCV screening, link-to-diagnosis, and link-to-treatment with direct-acting antiviral agents (DAA). The primary end-point was sustained virological response (SVR). RESULTS: Of 8291 registered residents who were invited as the target population, 7807 (94.2%) subjects received anti-HCV screening, with the average anti-HCV prevalence rate of 14.2% (1108/7807) (range among four areas: 11.8%-16.7%). The rate of link-to-diagnosis was 94.4% (1046/1108) of anti-HCV-positive subjects (range: 90.9%-100%) with an average HCV-viremic rate of 55.1% (576/1046) (range: 50.0%-64.3%). The link-to-treat rate was 94.4% (544/576) in HCV-viremic subjects (range from 92.7% to 97.2%). Overall, 523 (96.1%) patients achieved an SVR (range: 94.7%-97.6%). Eventually, the overall effectiveness was 80.7% (range: 74.6%-93.1%). The presence of hepatocellular carcinoma at baseline was the only factor associated with DAA failure. The DAA regimens were well-tolerated. CONCLUSION: The outreach decentralized community-based care system with DAA therapy was highly effective and safe in the achievement of HCV micro-elimination in the resource-constrained rural and remote regions, which could help us to tackle the disparity.

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.

Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.

Assembling bacteriophage T7 leading-strand replisome for structural investigation.

Replicative helicase and polymerase form the leading-strand replisome that unwinds parental DNA and performs continuous leading-strand DNA synthesis. Uncoupling of the helicase-polymerase complex results in replication stress, replication errors, and genome instability. Although numerous replisomes from different biological systems have been reconstituted and characterized, structural investigations of the leading-strand replisome complex are hindered by its large size and dynamics. We have determined the first replisome structure on a fork substrate with bacteriophage T7 replisome as a model system. Here, we summarized our protocols to prepare and characterize the coupled T7 replisome complex. Similar methods can potentially be applied for structural investigations of more complicated replisomes.

DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication.

DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular basis of the helicase-polymerase coupling is not fully understood. The recently elucidated T7 replisome structure suggests that the helicase and polymerase sandwich parental DNA and each enzyme pulls a daughter strand in opposite directions. Interestingly, the T7 polymerase, but not the helicase, carries the parental DNA with a positively charged cleft and stacks at the fork opening using a ß-hairpin loop. Here, we created and characterized T7 polymerases each with a perturbed ß-hairpin loop and positively charged cleft. Mutations on both structural elements significantly reduced the strand-displacement synthesis by T7 polymerase but had only a minor effect on DNA synthesis performed against a linear DNA substrate. Moreover, the aforementioned mutations eliminated synergistic helicase-polymerase binding and unwinding at the DNA fork and processive fork progressions. Thus, our data suggested that T7 polymerase plays a dominant role in helicase-polymerase coupling and replisome progression.

The orf virus (ORFV) protein OV20.0 interacts with the microprocessor complex subunit DGCR8 to regulate miRNA biogenesis and ORFV infection.

Cellular double-stranded RNA-binding proteins (DRBPs) play important roles in the regulation of innate immune responses and microRNA (miRNA) biogenesis. The current study aimed to understand whether OV20.0, a DRBP of orf virus (ORFV), is involved in cellular RNA biogenesis via association with host DRBPs. We found that OV20.0 interacts with DiGeorge syndrome critical region 8 (DGCR8), a subunit of the miRNA processor complex, and binds to primary- and precursor-miRNA. Additionally, OV20.0 regulates DGCR8 expression in multiple ways, including through interaction with the DGCR8 protein and binding to DGCR8 mRNA. Lastly, our data show that DGCR8 plays an antiviral role against ORFV infection, whereas it is beneficial for influenza virus propagation, indicating that the underlying mechanisms could be diverse among different viruses.

DNA Helicase-Polymerase Coupling in Bacteriophage DNA Replication.

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase-polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel-Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability.

Coronaviruses are able to establish persistence. However, how coronaviruses react to persistence and whether the selected viruses have altered their characteristics remain unclear. In this study, we found that the persistent infection of bovine coronavirus (BCoV), which is in the same genus as SARS-COV-2, led to alterations of genome structure, attenuation of gene expression, and the synthesis of subgenomic mRNA (sgmRNA) with a previously unidentified pattern. Subsequent analyses revealed that the altered genome structures were associated with the attenuation of gene expression. In addition, the genome structure at the 5' terminus and the cellular environment during the persistence were responsible for the sgmRNA synthesis, solving the previously unanswered question regarding the selection of transcription regulatory sequence for synthesis of BCoV sgmRNA 12.7. Although the BCoV variants (BCoV-p95) selected under the persistence replicated efficiently in cells without persistent infection, its pathogenicity was still lower than that of wild-type (wt) BCoV. Furthermore, in comparison with wt BCoV, the variant BCoV-p95 was not able to efficiently adapt to the challenges of alternative environments, suggesting wt BCoV is genetically robust. We anticipate that the findings derived from this fundamental research can contribute to the disease control and treatments against coronavirus infection including SARS-CoV-2.

The Impacts of Antivirals on the Coronavirus Genome Structure and Subsequent Pathogenicity, Virus Fitness and Antiviral Design.

With the global threat of SARS-CoV-2, much effort has been focused on treatment and disease control. However, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics are also unanswered questions with medical importance. To this end, bovine coronavirus (BCoV), which is in the same genus as SARS-CoV-2, was used as a test model and the findings were as follows. With the treatment of antiviral remdesivir, the selected BCoV variant with an altered genome structure developed resistance, but its pathogenicity was not increased in comparison to that of wild type (wt) BCoV. Under the selection pressure of innate immunity, the genome structure was also altered; however, neither resistance developed nor pathogenicity increased for the selected BCoV variant. Furthermore, both selected BCoV variants showed a better efficiency in adapting to alternative host cells than wt BCoV. In addition, the previously unidentified feature that the spike protein was a common target for mutations under different antiviral treatments might pose a problem for vaccine development because spike protein is a common target for antibody and vaccine designs. The findings derived from this fundamental research may contribute to the disease control and treatments against coronaviruses, including SARS-CoV-2.

Interaction of coronavirus nucleocapsid protein with the 5'- and 3'-ends of the coronavirus genome is involved in genome circularization and negative-strand RNA synthesis.

Synthesis of the negative-strand ((-)-strand) counterpart is the first step of coronavirus (CoV) replication; however, the detailed mechanism of the early event and the factors involved remain to be determined. Here, using bovine coronavirus (BCoV)-defective interfering (DI) RNA, we showed that (a) a poly(A) tail with a length of 15 nucleotides (nt) was sufficient to initiate efficient (-)-strand RNA synthesis and (b) substitution of the poly(A) tail with poly(U), (C) or (G) only slightly decreased the efficiency of (-)-strand synthesis. The findings indicate that in addition to the poly(A) tail, other factors acting in trans may also participate in (-)-strand synthesis. The BCoV nucleocapsid (N) protein, an RNA-binding protein, was therefore tested as a candidate. Based on dissociation constant (Kd ) values, it was found that the binding affinity between N protein, but not poly(A)-binding protein, and the 3'-terminal 55 nt plus a poly(A), poly(U), poly(C) or poly(G) tail correlates with the efficiency of (-)-strand synthesis. Such an association was also evidenced by the binding affinity between the N protein and 5'- and 3'-terminal cis-acting elements important for (-)-strand synthesis. Further analysis demonstrated that N protein can act as a bridge to facilitate interaction between the 5'- and 3'-ends of the CoV genome, leading to circularization of the genome. Together, the current study extends our understanding of the mechanism of CoV (-)-strand RNA synthesis through involvement of N protein and genome circularization and thus may explain why the addition of N protein in trans is required for efficient CoV replication.

Gene Variations in Cis-Acting Elements between the Taiwan and Prototype Strains of Porcine Epidemic Diarrhea Virus Alter Viral Gene Expression.

In 2013, the outbreak of porcine epidemic diarrhea (PED) in Taiwan caused serious economic losses. In this study, we examined whether the variations of the cis-acting elements between the porcine epidemic diarrhea virus (PEDV) Taiwan (TW) strain and the prototype strain CV777 alter gene expression. For this aim, we analyzed the variations of the cis-acting elements in the 5' and 3' untranslated regions (UTRs) between the PEDV TW, CV777, and other reference strains. We also determined the previously unidentified transcription regulatory sequence (TRS), a sequence motif required for coronavirus transcription, and found that a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS. Functional analyses using coronavirus defective interfering (DI) RNA revealed that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. The current data show for the first time the evolution of PEDV in terms of cis-acting elements and their effects on gene expression, and thus may contribute to our understanding of recent PED outbreaks worldwide.

Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell.

In the present study, we investigated the roles of interactions among the poly(A) tail, coronavirus nucleocapsid (N) protein, and poly(A)-binding protein (PABP) in the regulation of coronavirus gene expression. Through dissociation constant (Kd ) comparison, we found that the coronavirus N protein can bind to the poly(A) tail with high affinity, establishing N protein as a PABP. A subsequent analysis with UV cross-linking and immunoprecipitation revealed that the N protein is able to bind to the poly(A) tail in infected cells. Further examination demonstrated that poly(A) tail binding by the N protein negatively regulates translation of coronaviral RNA and host mRNA both in vitro and in cells. Although the N protein can interact with PABP and eukaryotic initiation factor 4G (eIF4G), the poor interaction efficiency between the poly(A)-bound N protein and eIF4E may explain the observed decreased translation efficiency. In addition to interaction with translation factor eIF4G, the N protein is able to interact with coronavirus nonstructural protein 9 (nsp9), a replicase protein required for replication. The study demonstrates interactions among the poly(A) tail, N protein, and PABP both in vitro and in infected cells. Of the interactions, binding of the poly(A) tail to N protein decreases the interaction efficiency between the poly(A) tail and eIF4E, leading to translation inhibition. The poly(A)-dependent translation inhibition by N protein has not been previously demonstrated and thus extends our understanding of coronavirus gene expression.IMPORTANCE Gene expression in coronavirus is a complicated and dynamic process. In this study, we demonstrated that coronavirus N protein is able to bind to the poly(A) tail with high affinity, establishing N protein as a PABP. We also show how the interplay between coronavirus 3' poly(A) tail, PABP, and N protein regulates gene expression of the coronavirus and host cell. Of the interactions, poly(A) tail binding by the N protein negatively regulates translation, and to our knowledge, this inhibition of translation by binding of the N protein to poly(A) tail has not been previously studied. Accordingly, the study provides fundamental molecular details regarding coronavirus infection and expands our knowledge of coronavirus gene expression.

Characterization of the Role of Hexamer AGUAAA and Poly(A) Tail in Coronavirus Polyadenylation.

Similar to eukaryotic mRNA, the positive-strand coronavirus genome of ~30 kilobases is 5'-capped and 3'-polyadenylated. It has been demonstrated that the length of the coronaviral poly(A) tail is not static but regulated during infection; however, little is known regarding the factors involved in coronaviral polyadenylation and its regulation. Here, we show that during infection, the level of coronavirus poly(A) tail lengthening depends on the initial length upon infection and that the minimum length to initiate lengthening may lie between 5 and 9 nucleotides. By mutagenesis analysis, it was found that (i) the hexamer AGUAAA and poly(A) tail are two important elements responsible for synthesis of the coronavirus poly(A) tail and may function in concert to accomplish polyadenylation and (ii) the function of the hexamer AGUAAA in coronaviral polyadenylation is position dependent. Based on these findings, we propose a process for how the coronaviral poly(A) tail is synthesized and undergoes variation. Our results provide the first genetic evidence to gain insight into coronaviral polyadenylation.

Dynamic postural control during trunk bending and reaching in healthy adults and stroke patients.

OBJECTIVES: Postural stability is often task-demand-dependent and often impaired in stroke patients. The purposes of this study were (1) to compare the difference in the center of pressure measures during forward bending of the trunk and reaching on postural actions between normal subjects and stroke patients and (2) to examine the effects of task demands on the center of pressure measures in both groups. DESIGN: Thirteen normal subjects and 23 stroke patients performed two trials of forward bending of the trunk and reaching for each of the targets at six locations at floor level while standing on a 0.5-m-long pressure mat. Center of pressure excursion, center of pressure average velocity, and bilateral limb weight-bearing ratios characterized the postural actions. Mixed-design analysis of variance was used. RESULTS: A significant target location by group interaction effects was found for all variables except the center of pressure average velocity. All variables except center of pressure average velocity showed significant group differences. The center of pressure excursion and bilateral limb weight-bearing ratio were smaller, and the center of pressure average velocity was larger in stroke patients than in normal subjects. The effects of target location on variables measured were prominent in stroke patients. The posturography of stroke patients was less regular than that of normal subjects. Stroke patients tended to avoid shifting their center of pressure toward the affected side, even when highly necessary. CONCLUSIONS: The difference of postural actions between groups depended on task demands. Small center of pressure displacement and fast center of pressure velocity characterized decreased adaptive postural actions. The amount of center of pressure shift and center of pressure velocity were also dependent on the task demands for both groups.

Physiological anterior laxity in healthy young females: the effect of knee hyperextension and dominance.

Female athletes are more likely to sustain an anterior cruciate ligament (ACL) injury. Knee laxity, hyperextension and limb dominance have been suggested as possible factors contributing to the knee injury. The aims of this study were to investigate the physiological anterior knee laxity between the dominant and non-dominant limb and in healthy young females with and without hyperextension knees. Forty-two healthy young females, 21 with hyperextension knees, were recruited voluntarily for this study. The subjects were tested with KT-2000 knee ligament arthrometer at both knees with flexion 30 degrees to obtain the anterior tibial displacements at loadings of 45, 67, 89 and 134 N. The initial and terminal stiffnesses were further calculated and analyzed to demonstrate the differences in the characteristics of knee laxity between limbs and groups. The results showed that there was no significant displacement difference between hyperextension and non-hyperextension groups. However, different physiological anterior laxities were illustrated for the different limbs and groups. The non-dominant side of the hyperextension group had significantly smaller terminal stiffness than that of the non-hyperextension group. The dominant side of the hyperextension group had larger laxity than the non-dominant side in the higher loading conditions. These findings may explain hyperextension knees are at greater risk of sustaining an ACL injury.

Isokinetic characteristics of shoulder rotators in patients with adhesive capsulitis.

OBJECTIVE: To demonstrate the muscle strength characteristics of shoulder internal and external rotators and the effects of isokinetic exercise on muscle activity in patients with adhesive capsulitis. DESIGN: Cohort study with control subjects. PARTICIPANTS: Eight patients with adhesive capsulitis and 8 controls. METHODS: Maximal isometric and isokinetic strength tests of shoulder internal and external rotators in the scapular plane were carried out. Muscle activities of the rotators were recorded in resting and during maximal isometrics. Muscle strength variables (peak torque, total work and power) and myoelectric variables (resting root-mean-square amplitude pre- and post-tests and the external-internal rotator co-activity in resting and during isometric contractions) were recorded. A mixed repeated-measure analysis of variance test was used to examine the within-group and between-group differences. RESULTS: For affected shoulders, smaller isometric average torque of internal rotators and high-speed peak torque, total work and power of external rotator were observed. The external/internal rotator ratio of peak torque in high-speed testing also exhibited significant decrease. The myoelectric variables showed no significant changes. CONCLUSION: High-speed external rotator strength and isometric internal rotator strength of the affected shoulders were decreased significantly. Isokinetic exercise may not increase the resting muscle activities and co-activity. These results provide a reference in planning muscle strengthening programmes and goals for these patients.