A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing Group II Intron.

Zika virus (ZIKV) is a mosquito-borne flavivirus of the Flaviviridae family first isolated from a sentinel monkey in the Zika Forest, Uganda, in 1947. Since 2007, the virus has had a vast geographic expansion that extended to the Americas in 2015, leading to a series of large outbreaks. Although mainly transmitted by the bite of Aedes mosquitoes, human infection of ZIKV can also happen through unconventional routes such as sexual intercourse and, more importantly, vertical transmission. The genome of ZIKV is a single-stranded, positive-sense RNA molecule about 11 kb in length. The genome contains a single opening reading frame (ORF) flanked by highly structured 5' and 3' untranslated regions. To understand the mechanisms about ZIKV replication, transmission, and pathogenesis, reverse genetic tools are of great importance. In this chapter, a novel system is described for the generation and manipulation of a ZIKV infectious clone stabilized by a self-splicing group II intron, a mobile element with ribozyme activity. The intron can be spliced in vitro, and thus full-length vRNA can be prepared allowing virus genome manipulation required for further studies.

Specialized cis-Acting RNA Elements Balance Genome Cyclization to Ensure Efficient Replication of Yellow Fever Virus.

Genome cyclization is essential for viral RNA (vRNA) replication of the vertebrate-infecting flaviviruses, and yet its regulatory mechanisms are not fully understood. Yellow fever virus (YFV) is a notorious pathogenic flavivirus. Here, we demonstrated that a group of cis-acting RNA elements in YFV balance genome cyclization to govern efficient vRNA replication. It was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) is conserved in the YFV clade and is important for efficient YFV propagation. By using two different replicon systems, we found that the function of the DCS-HP is determined primarily by its secondary structure and, to a lesser extent, by its base-pair composition. By combining in vitro RNA binding and chemical probing assays, we found that the DCS-HP orchestrates the balance of genome cyclization through two different mechanisms, as follows: the DCS-HP assists the correct folding of the 5' end in a linear vRNA to promote genome cyclization, and it also limits the overstabilization of the circular form through a potential crowding effect, which is influenced by the size and shape of the DCS-HP structure. We also provided evidence that an A-rich sequence downstream of the DCS-HP enhances vRNA replication and contributes to the regulation of genome cyclization. Interestingly, diversified regulatory mechanisms of genome cyclization, involving both the downstream of the 5'-cyclization sequence (CS) and the upstream of the 3'-CS elements, were identified among different subgroups of the mosquito-borne flaviviruses. In summary, our work highlighted how YFV precisely controls the balance of genome cyclization to ensure viral replication. IMPORTANCE Yellow fever virus (YFV), the prototype of the Flavivirus genus, can cause devastating yellow fever disease. Although it is preventable by vaccination, there are still tens of thousands of yellow fever cases per year, and no approved antiviral medicine is available. However, the understandings about the regulatory mechanisms of YFV replication are obscure. In this study, by a combination of bioinformatics, reverse genetics, and biochemical approaches, it was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) promotes efficient YFV replication by modulating the conformational balance of viral RNA. Interestingly, we found specialized combinations for the downstream of the 5'-cyclization sequence (CS) and upstream of the 3'-CS elements in different groups of the mosquito-borne flaviviruses. Moreover, possible evolutionary relationships among the various downstream of the 5'-CS elements were implied. This work highlighted the complexity of RNA-based regulatory mechanisms in the flaviviruses and will facilitate the design of RNA structure-targeted antiviral therapies.

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Development of a Bicistronic Yellow Fever Live Attenuated Vaccine with Reduced Neurovirulence and Viscerotropism.

The yellow fever (YF) live attenuated vaccine strain 17D (termed 17D) has been widely used for the prevention and control of YF disease. However, 17D retains significant neurovirulence and viscerotropism in mice, which is probably linked to the increased occurrences of serious adverse events following 17D vaccination. Thus, the development of an updated version of the YF vaccine with an improved safety profile is of high priority. Here, we generated a viable bicistronic YF virus (YFV) by incorporating the internal ribosome entry site (IRES) from Encephalomyocarditis virus into an infectious clone of YFV 17D. The resulting recombinant virus, 17D-IRES, exhibited similar replication efficiency to its parental virus (17D) in mammalian cell lines, while it was highly restricted in mosquito cells. Serial passage of 17D-IRES in BHK-21 cells showed good genetic stability. More importantly, in comparison with the parental 17D, 17D-IRES displayed significantly decreased mouse neurovirulence and viscerotropism in type I interferon (IFN)-signaling-deficient and immunocompetent mouse models. Interestingly, 17D-IRES showed enhanced sensitivity to type I IFN compared with 17D. Moreover, immunization with 17D-IRES provided solid protection for mice against a lethal challenge with YFV. These preclinical data support further development of 17D-IRES as an updated version for the approved YF vaccine. This IRES-based attenuation strategy could be also applied to the design of live attenuated vaccines against other mosquito-borne flaviviruses. IMPORTANCE Yellow fever (YF) continually spreads and causes epidemics around the world, posing a great threat to human health. The YF live attenuated vaccine 17D is considered the most efficient vaccine available and helps to successfully control disease epidemics. However, side effects may occur after vaccination, such as viscerotropic disease (YEL-AVD) and neurotropic adverse disease (YEL-AND). Thus, there is an urgent need for a safer YF vaccine. Here, an IRES strategy was employed, and a bicistronic YFV was successfully developed (named 17D-IRES). 17D-IRES showed effective replication and genetic stability in vitro and high attenuation in vivo. Importantly, 17D-IRES induced humoral and cellular immune responses and conferred full protection against lethal YFV challenge. Our study provides data suggesting that 17D-IRES, with its prominent advantages, could be a vaccine candidate against YF. Moreover, this IRES-based bicistronic technology platform represents a promising strategy for developing other live attenuated vaccines against emerging viruses.

[Research progress of full endoscopic transforaminal lumbar interbody fusion].

Full endoscopic transforaminal lumbar interbody fusion has been used widely in the field of minimally invasive spine surgery in recent years. This paper briefly introduces the development history, technical points, indications, curative effects and complications. Authors believe that the full endoscopic transforaminal lumbar interbody fusion has the same clinical effects as traditional surgery, and can effectively reduce tissue damage and intraoperative bleeding, reduce the incidence of postoperative low back pain, shorten the time to get out of bed, and reduce the average hospitalization time. However, it is still necessary to improve the long-term follow up in order to further evaluate the effectiveness and safety of the procedure.

Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D.

Yellow fever virus (YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans. Despite the availability of an effective vaccine, little is known about the replication mechanism of YFV, and there are still no available specific anti-YFV medicines. Herein, by introducing the Renilla luciferase gene (Rluc) into an infectious clone of YFV vaccine strain 17D, we generated a recombinant virus 17D-Rluc.2A via reverse genetics approaches. The 17D-Rluc.2A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain. Importantly, the reporter luciferase was efficiently expressed in 17D-Rluc.2A-infected mammalian and mosquito cells, and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction. Furthermore, by a combination of the 17D-Rluc.2A reporter virus and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) technology, the conserved 5'-SLA element was shown to be essential for YFV replication, highlighting the capability of 17D-Rluc.2A in the investigation of YFV replication. At last, we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17D-Rluc.2A-infected cells, demonstrating its potential application in the evaluation of anti-viral medicines. Taken together, the 17D-Rluc.2A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.

Visualization of yellow fever virus infection in mice using a bioluminescent reporter virus.

Yellow fever virus (YFV) is a re-emerging flavivirus, which can lead to severe clinical manifestations and high mortality, with no specific antiviral therapies available. The live-attenuated yellow fever vaccine 17D (YF17D) has been widely used for over eighty years. However, the emergence of yellow fever vaccine-associated viscerotropic disease (YFL-AVD) and yellow fever vaccine-associated neurotropic disease (YFL-AND) raised non-negligible concerns. Additionally, the attenuation mechanism of YF17D is still unclear. Thus, the development of convenient models is crucial to understand the mechanisms behind YF17D attenuation and its adverse effects. In this work, we generated a reporter YF17D expressing nano-luciferase (NLuc). In vitro and in vivo characterization demonstrated that the NLuc-YF17D shared similar biological properties with its parental strain and the NLuc activity can reflect viral infectivity reliably. Combined with in vivo bioluminescence imaging, a series of mice models of YF17D infection was established, which will be useful for the evaluation of antiviral medicines and novel vaccine candidates. Especially, we demonstrated that intraperitoneally (i.p.) infection of NLuc-YF17D in type I interferon receptor-deficient mice A129 resulted in outcomes resembling YEL-AVD and YEL-AND, evidenced by viral replication in multiple organs and invasion of the central neuronal system. Finally, in vitro and in vivo assays based on this reporter virus were established to evaluate the antiviral activities of validated antiviral agents. In conclusion, the bioluminescent reporter virus described herein provides a powerful platform to study YF17D attenuation and vaccine-associated diseases as well as to develop novel countermeasures against YFV.

In vitro Studies and Clinical Observations Imply a Synergistic Effect Between Epstein-Barr Virus and Dengue Virus Infection.

Dengue virus (DENV) infection can lead to a complex spectrum of clinical outcomes, ranging from asymptomatic infection to life-threatening severe dengue. The reasons for thus drastically varying manifestations of the disease remain an enigma. Herein, we reported an original discovery of the synergistic effect between preexisting Epstein-Barr virus (EBV) infection and DENV superinfection in vitro and of a strong correlation of these two viruses in the clinical samples from dengue patients. We showed that (I) DENV-2 infection of an EBV-positive cell line (EBV + Akata cell) reactivated EBV, and it could be blocked by wortmannin treatment. (II) Examination of human peripheral blood mononuclear cell (PBMC) samples from dengue patients revealed significantly elevated cell-associated EBV DNA copy number at the time of hospitalization vs. at the time of disease recovery in most individuals. (III) EBV infection promoted DENV propagation in both EBV-hosting B cells and indirectly in THP-1 cells, supported by the following evidence: (A) EBV + Akata cells were more permissive to DENV-2 infection compared with Akata cells harboring no EBV virus (EBV- Akata cells). (B) Low-molecular weight fraction secreted from EBV + Akata cells could enhance DENV-2 propagation in monocytic THP-1 cells. (C) While reactivation of EBV in EBV + Akata cells further increased DENV-2 yield from this cell line, pharmacological inhibition of EBV replication by acyclovir had the opposite effect. To our knowledge, this is the first investigation demonstrating a positive correlation between EBV and DENV in vitro and in human biospecimens.

Horizontal Rafting Plate for Treatment of the Tibial Plateau Fracture.

OBJECTIVE: This study aimed to investigate the value of a horizontal rafting plate in treating tibial plateau fractures. METHODS: The data of 24 patients in whom a horizontal rafting plate was used to treat a tibial plateau fracture between October 2014 and January 2018 were retrospectively analyzed, including 16 males and 8 females, aged 21-63 years old, with an average of 40 ± 14.68 years. The fractures included 13 in the left knee and 11 in the right knee. The places where the horizontal rafting plate were used included the anterior margin of tibia, anterolateral tibia, and posterolateral tibia. All cases were followed up for 12-24 months, with an average follow-up of 17.5 ± 5.0 months. At the last follow-up, the Rasmussen radiological criteria were used to evaluate the effect of fracture reduction and fixation. The knee joint function was evaluated using the Rasmussen functional score. Computed tomography (CT) scanning and three-dimensional reconstruction were performed preoperatively and postoperatively, with the quality of reduction of the fractured articular surface clarified by the final follow-up. The flexion and extension abilities of the knee joint were also measured in the postoperative follow-up. RESULTS: Preoperative CT scanning showed that the gap of the tibial plateau was 8.00 ± 1.40 (5-24) mm. The heights of the fracture of the articular surface at all three sites during the final follow-ups were significantly different from the height before the surgery (P < 0.05). The vertical distance between the articular line and the highest point of the articular surface after reduction was 0.17 ± 0.05 mm. Anatomic reductions were obtained in 24 patients. The Rasmussen functional score after surgeries was 27.25 ± 0.94 points. Bony union was achieved in all the patients. According to the Rasmussen radiological criteria, the scores during the last follow-up were as follows: the total score was 13-18 points, with an average of 16.00 ± 1.72 points; the scores were excellent in 17 cases and good in seven cases. Therefore, 100% of results were excellent or good. No infection or fracture nonunion was found. CONCLUSION: Using a horizontal plate can be an effective method for treating special types of fractures of the tibial plateau, including the anterior margin and anterolateral and posterolateral tibial plateau, with satisfactory treatment efficacy.

Surgical Strategy for Anterior Tibial Plateau Fractures in Hyperextension Knee Injuries.

OBJECTIVE: The aim of the present study was to summarize the clinical characteristics, treatment strategies, and clinical results for anterior tibial plateau fractures caused by hyperextension injuries. METHODS: We performed a retrospective analysis of 26 cases of anterior tibial plateau fractures that were treated with open reduction and internal fixation from January 2016 to December 2019, including 16 men and 10 women, aged 26-68 years old, with an average age of 47 ± 12.5 years. According to the three-column theory classification, there were 16 cases of single-column fractures (9 cases of anteromedial fractures and 7 cases of anterolateral fractures), 3 cases of two-column fractures (anteromedial + anterolateral fractures), and 7 cases of three-column fractures. Options for the surgical approach included anteromedial, anterolateral, modified anterior median, and anterolateral + posteromedial incision. The implants included a T-shaped plate, an L-shaped plate, a horizontal plate, and a TomoFix plate. The surgical approach and fixation method were selected based on the characteristics of the anterior tibial fracture. The Rasmussen radiological criteria were used to evaluate the effects of fracture reduction and fixation. The knee joint function was evaluated using the knee function evaluation criteria of the Hospital for Special Surgery. Medial and lateral stress tests, the Lachman test, and the pivot shift test were used to evaluate the stability of the knee joint. The range of knee motion was recorded. RESULTS: All cases were followed up for 12-24 months, with an average follow up of 15.7 months. The operation time was (148 ± 42) min; the intraoperative blood loss was (150 ± 50) mL. A total of 22 cases were anatomically reduced and 4 cases were well-reduced, and the compression reduction rate was 100%. According to the Rasmussen radiology scoring, 17 cases were excellent and 9 cases were good. The excellent and good rate was 100%. The fracture healing time was 3.3 months. There is no difference in fracture healing time for different fracture types. Both the Lachman and pivot shift test findings were normal in 24 patients and nearly normal in 2 patients. The posterior drawer test was normal in 25 patients and close to normal in 1 patient. The varus stress test was normal in 24 patients and nearly normal in 2 patients, while the valgus stress test was normal in 23 patients, nearly normal in 2 patients, and abnormal in 1 patient. The range of motion (ROM) was 100°-137°, with an average of 125° ± 11.7°. The Hospital for Special Surgery (HSS) knee score at the last follow up was 79-98 points, with an average of 87.54 ± 8.36 points; the results were excellent in 21 cases and good in 5 cases. Therefore, 100% of results were excellent or good. Two cases had superficial wound infections after the operation. The recovery of 2 patients with common peroneal nerve injury was poor. CONCLUSION: The appropriate surgical approach and fixation method were performed according to the different positions of the anterior tibial fracture and satisfactory results were obtained after surgery.

Repeated subarachnoid administrations of allogeneic human umbilical cord mesenchymal stem cells for spinal cord injury: a phase 1/2 pilot study.

BACKGROUND AIMS: Stem cell transplantation is a potential treatment for intractable spinal cord injury (SCI), and allogeneic human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising candidate because of the advantages of immune privilege, paracrine effect, immunomodulatory function, convenient collection procedure and little ethical concern, and there is an urgent need to develop a safe and effective protocol regarding their clinical application. METHODS: A prospective, single-center, single-arm study in which subjects received four subarachnoid transplantations of hUC-MSCs (1 × 106 cells/kg) monthly and were seen in follow-up four times (1, 3, 6 and 12 months after final administration) was conducted. At each scheduled time point, safety and efficacy indicators were collected and analyzed accordingly. Adverse events (AEs) were used as a safety indicator. American Spinal Injury Association (ASIA) and SCI Functional Rating Scale of the International Association of Neurorestoratology (IANR-SCIFRS) total scores at the fourth follow-up were determined as primary efficacy outcomes, whereas these two indicators at the remaining time points as well as scores of pinprick, light touch, motor and sphincter, muscle spasticity and spasm, autonomic system, bladder and bowel functions, residual urine volume (RUV) and magnetic resonance imaging (MRI) were secondary efficacy outcomes. Subgroup analysis of primary efficacy indicators was also performed. RESULTS: Safety and efficacy assessments were performed on 102 and 41 subjects, respectively. Mild AEs involving fever (14.1%), headache (4.2%), transient increase in muscle tension (1.6%) and dizziness (1.3%) were observed following hUC-MSC transplantation and resolved thoroughly after conservative treatments. There was no serious AE. ASIA and IANR-SCIFRS total scores revealed statistical increases when compared with the baselines at different time points during the study, mainly reflected in the improvement of pinprick, light touch, motor and sphincter scores. Moreover, subjects showed a continuous and remarkable decrease in muscle spasticity. Regarding muscle spasm, autonomic system, bladder and bowel functions, RUV and MRI, data/imaging at final follow-up showed significant improvements compared with those at first collection. Subgroup analysis found that hUC-MSC transplantation improved neurological functions regardless of injury characteristics, including level, severity and chronicity. CONCLUSIONS: The authors' present protocol demonstrates that intrathecal administration of' allogeneic hUC-MSCs at a dose of 106 cells/kg once a month for 4 months is safe and effective and leads to significant improvement in neurological dysfunction and recovery of quality of life.

Structure and function of cis-acting RNA elements of flavivirus.

The genus Flavivirus is a group of single-stranded, positive-sense RNA viruses that includes numerous human pathogens with global impact, such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Zika virus (ZIKV). The approximately 11-kilobase genome is flanked by highly structured untranslated regions (UTRs), which contain various cis-acting RNA elements with unique structures and functions. Moreover, local RNA elements circularize the genome non-covalently through long-range interactions. Interestingly, many flavivirus cis-acting RNA elements contain group-specific motifs or are specific for the given phylogenetic groups, suggesting their potential association with flavivirus evolution and diversification. In this review, we summarize recent advances about the structure and function of cis-acting RNA elements in flavivirus genomes and highlight the potential implications for flavivirus evolution. Finally, the scientific questions remained to be answered in the field are also discussed.

Zika NS1-induced ER remodeling is essential for viral replication.

Zika virus (ZIKV), a recently emerged member of the flavivirus family, forms replication compartments at the ER during its lifecycle. The proteins that are responsible for the biogenesis of replication compartments are not well defined. Here, we show that Zika nonstructural protein 1 (NS1)-induced ER remodeling is essential for viral replication. NS1 expressed in the ER lumen induced ER perinuclear aggregation with an ultrastructure resembling that of the replication compartment. Data from model membrane system indicated that the membrane-binding and membrane-remodeling properties of NS1 depend on its hydrophobic insertion into the membrane. These findings demonstrate that NS1 plays a crucial role in flavivirus replication compartment formation by remodeling the ER structure.

Surgical Treatment of Lateral Tibial Plateau Fractures Involving the Posterolateral Column.

OBJECTIVE: To summarize the indications and the clinical effects of a transfibular neck osteotomy approach and a combined anterolateral and posterolateral approach in the treatment of fractures of the lateral tibial plateau involving the posterolateral column. METHODS: Eleven patients with lateral tibial plateau fractures were included in the present study. The fractures were Schatzker type II or lateral platform fractures involving posterolateral column. The anterolateral combined posterolateral approach (lateral + posterolateral locking plate fixation) was applied in 7 patients and 4 patients underwent transfibular neck osteotomy (lateral + posterolateral locking plate fixation + 1/4 tubular plate edge fixation, fibular osteotomy with Kirschner wire tension band fixation, and hollow nail fixation for upper tibiofibular joint). All cases were followed up for 12-24 months, with an average follow-up of 17.5 ± 5.0 months. At the last followup, the Rasmussen radiological criteria were used to evaluate the effect of fracture reduction and fixation. The knee joint function was evaluated using the knee function evaluation criteria of the Hospital for Special Surgery (HSS). The Lachman test and the pivot-shift test were used to evaluate the anterior and posterior and rotational stability of the knee joint. The range of knee motion was recorded. RESULTS: Bone healing was achieved in all patients with fractures treated with a transfibular neck osteotomy approach and a combined anterolateral and posterolateral approach. At the last follow-up, both the Lachman test and the pivot-shift test results were negative. All patients had complete knee extension. For the combined anterolateral and posterolateral approach, the knee flexion angle was 110°-130°, with an average of 122.86° ± 7.56°. For the transfibular neck osteotomy approach, the knee flexion angle was 115°-130°, with an average of 120.00° ± 7.07°. For the patients in which the combined anterolateral and posterolateral approach was used, the Rasmussen score was 12-18 points, with an average of 16.00 ± 2.56 points. The results were excellent in 4 cases and good in 3 cases; therefore, 100% of results were excellent or good. For patients in which the transfibular neck osteotomy approach was used, the Rasmussen score was 10-18 points, with an average of 15.25 ± 3.77 points. The results were excellent in 2 cases, good in 1 case, and acceptable in 1 case; therefore, 75% of results were excellent or good. The HSS score for the combined anterolateral and posterolateral approach was 76-98 points, with an average of 88.43 ± 7.55 points. The results were excellent in 5 cases and good in 2 cases; therefore, 100% of results were excellent or good. The HSS score for the transfibular neck osteotomy approach was 74-96 points, with an average of 87.25 ± 9.43 points. The results were excellent in 3 cases and good in 1 case; therefore, 100% of results were excellent or good. There were no significant differences in operation time, surgical blood loss, fracture healing time, postoperative imaging score, and knee function evaluation between the two approaches. One patient who underwent transfibular neck osteotomy had a 3-mm step that gradually appeared, but no significant abnormalities were found in the width of the platform and the lower limb force line. One patient in whom the combined anterolateral and posterolateral approach was used showed numbness in the common peroneal nerve. No common peroneal nerve injury occurred through the transfibular neck osteotomy approach. CONCLUSIONS: The anterolateral combined posterolateral approach and the transfibular neck osteotomy approach are effective in the surgical treatment of lateral tibial plateau fractures involving the posterolateral column. However, the transfibular neck osteotomy approach is more suitable for the posterolateral plateau articular surface damaged with bone separation and displacement, deep collapse, cases involving a large range of the posterolateral column, especially fractures of the lateral tibial plateau in the upper tibiofibular syndesmosis area of the line connecting the anterior and posterior margin of the fibular head to the midpoint of the plateau.

Long non-coding subgenomic flavivirus RNAs have extended 3D structures and are flexible in solution.

Most mosquito-borne flaviviruses, including Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), produce long non-coding subgenomic RNAs (sfRNAs) in infected cells that link to pathogenicity and immune evasion. Until now, the structural characterization of these lncRNAs remains limited. Here, we studied the 3D structures of individual and combined subdomains of sfRNAs, and visualized the accessible 3D conformational spaces of complete sfRNAs from DENV2, ZIKV, and WNV by small angle X-ray scattering (SAXS) and computational modeling. The individual xrRNA1s and xrRNA2s adopt similar structures in solution as the crystal structure of ZIKV xrRNA1, and all xrRNA1-2s form compact structures with reduced flexibility. While the DB12 of DENV2 is extended, the DB12s of ZIKV and WNV are compact due to the formation of intertwined double pseudoknots. All 3' stem-loops (3'SLs) share similar rod-like structures. Complete sfRNAs are extended and sample a large conformational space in solution. Our work not only provides structural insight into the function of flavivirus sfRNAs, but also highlights strategies of visualizing other lncRNAs in solution by SAXS and computational methods.

Less Vertebral Bone Mass after Treatment with Macitentan in Mice: A Pilot Study.

PURPOSE: Blood vessels and skeleton interact together. Endothelin-1 is a potent vasoconstrictor and also has an effect on bone metabolism. The dual antagonist to both endothelin-1 type A and B receptors, Macitentan, has been approved for clinical management of pulmonary arterial hypertension while little is known about the secondary effect of the drug on spine. We aimed to answer how vertebral bone mass responded to Macitentan treatment in mice. METHODS: Sixteen male balb/c mice at 6 months were randomly assigned into 2 groups. Vehicle and Macitentan were administrated via intraperitoneal injection to Control group and Treatment group, respectively, for 4 months. At sacrifice, plasma endothelin-1 was evaluated with ELISA and vertebral bone mass was evaluated with Microcomputed Tomography and histological analysis. RESULTS: We found higher plasma endothelin-1 level (p<0.01) and less vertebral bone mass (p<0.05) in Treatment group compared to controls. Moreover, less osteoblasts and more osteoclasts were observed in the vertebral trabecular bone in the Treatment group compared to controls, by immunohistochemistry of the cell-specific markers. CONCLUSIONS: Treatment with Macitentan is associated with significant lower vertebral bone mass and therefore the secondary effect of dual antagonists to endothelin-1 receptors on the skeleton should be monitored and investigated in clinical practice. Both osteoblasts and osteoclasts may be involved while the molecular mechanism needs to be further explored.

The evolution of Zika virus from Asia to the Americas.

Zika virus (ZIKV) was once considered an obscure member of the large and diverse family of mosquito-borne flaviviruses, and human infections with ZIKV were thought to be sporadic, with mild and self-limiting symptoms. The large-scale ZIKV epidemics in the Americas and the unexpected uncovering of a link to congenital birth defects escalated ZIKV infections to the status of a global public health emergency. Recent studies that combined reverse genetics with modelling in multiple systems have provided evidence that ZIKV has acquired additional amino acid substitutions at the same time as congenital Zika syndrome and other birth defects were detected. In this Progress article, we summarize the evolution of ZIKV during its spread from Asia to the Americas and discuss potential links to pathogenesis.

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

Microendoscopy-Assisted Minimally Invasive Versus Open Transforaminal Lumbar Interbody Fusion for Lumbar Degenerative Diseases: 5-Year Outcomes.

OBJECTIVE: We sought to evaluate 5-year outcomes between microendoscopy-assisted minimally invasive (MIS) and open transforaminal lumbar interbody fusion (TLIF). METHODS: Sixty single-level MIS and open surgeries were performed (30 patients in either group). Perioperative parameters including operative duration, intraoperative estimated blood loss, fluoroscopy time, postoperative analgesic usage, ambulatory time, and complications were recorded. Visual analog scale (back and leg), Japanese Orthopaedics Association score, and Oswestry Disability Index were obtained. Finally, self-evaluation of surgical outcomes (modified MacNab criteria), interbody fusion rate (Bridwell grade 1), and prevalence of adjacent segment degeneration were assessed. RESULTS: Intraoperative estimated blood loss and postoperative analgesia usage were reduced in the MIS group, and patients undergoing microendoscopy-assisted MIS-TLIF ambulated earlier than those receiving open TLIF postoperatively. Nevertheless, surgical duration and fluoroscopy time were prolonged in the MIS group. Complication incidences were similar in both groups. Visual analog scale (back and leg), Japanese Orthopaedics Association, and Oswestry Disability Index were improved at 1 month, 2 years, and 5 years postoperatively in both groups when compared with preoperative scores. Significant improvements in these scores were found in the MIS group at 1 month postoperatively, while at 2 years and 5 years postoperatively, both groups revealed comparable aforementioned scores. Excellent and perfect scale rating, interbody fusion rate, and adjacent segment degeneration prevalence between the groups were almost similar. CONCLUSIONS: Microendoscopy-assisted MIS-TLIF is comparable with open TLIF in terms of 5-year outcomes with additional benefits of reduced intraoperative iatrogenic injury, decreased initial pain, minimized activity restrictions, and accelerated ambulation recovery after surgery.