Total Synthesis of Puberuline C.

Puberuline C (1) is an architecturally complex C19-diterpenoid alkaloid with a unique ring fusion pattern. The 6/7/5/6/6/6-membered rings (ABCDEF-rings) contain one tertiary amine and six oxygen functionalities, and possess 12 contiguously aligned stereocenters, three of which are quaternary. These structural features of 1 make its chemical construction exceptionally challenging. Here, we disclose the first total synthesis of 1. The synthesis was accomplished from 2-cyclohexenone (9) by integrating radical cascade and Mukaiyama aldol reactions as the key transformations. A double Mannich reaction fused the A- and E-rings, and Sonogashira coupling attached the C-ring, efficiently leading to ACE-rings with the requisite 19 carbons of 1. The chemically stable tertiary chloride of the ACE-ring structure was then transformed to the corresponding bridgehead radical, which participated in the simultaneous cyclization of the B- and F-rings via a highly organized radical cascade process. This unusual step installed five contiguous stereocenters, including two quaternary carbons, without damaging the preexisting multiple polar functionalities. Subsequently, the intramolecular Mukaiyama aldol reaction between silyl enol ether and acetal was realized by applying a combination of SnCl4 and ZnCl2, forging the last remaining D-ring of the hexacycle. Finally, 3 was elaborated into 1 through regio- and stereoselective functionalizations of the BCD-rings. Our novel radical-based strategy achieved the total synthesis of 1 in 32 total steps from simple 9, demonstrating the power of the radical cascade reaction to streamline the assembly of highly complex molecules.

Construction of Pentacyclic Limonoid Skeletons via Radical Cascade Reactions.

Limonoids 1 and 2 share a 6/6/6/5-membered ABCD-ring system and a six-membered oxacycle and differ in their C9-stereochemistries. A new radical-based strategy was devised to construct the pentacyclic skeletons of 1 and 2. An oxacycle-fused A-ring and enyne fragments were coupled to produce radical precursors 4a-4c with different C7-oxygen functionalities. The bridgehead tertiary bromide of 4a-4c participated in a radical cascade reaction with the three unsaturated bonds to cyclize the C9-diastereomeric BCD-rings.

Protective efficacy of recombinant Toxoplasma gondii dense granule protein 15 against toxoplasmosis in C57BL/6 mice.

Toxoplasma gondii is a pervasive protozoan parasite that is responsible for significant zoonoses. A wide array of vaccines using different effector molecules of T. gondii have been studied worldwide to control toxoplasmosis. None of the existing vaccines are sufficiently effective to confer protective immunity. Among the different Toxoplasma-derived effector molecules, T. gondii dense granule protein 15 from the type II strain (GRA15 (II)) was recently characterized as an immunomodulatory molecule that induced host immunity via NF-κB. Therefore, we assessed the immunostimulatory and protective efficacy of recombinant GRA15 (II) (rGRA15) against T. gondii infection in a C57BL/6 mouse model. We observed that rGRA15 treatment increased the production of IL-12p40 from mouse peritoneal macrophages in vitro. Immunization of mice with rGRA15 induced the production of anti-TgGRA15-specific IgG, IgG1 and IgG2c antibodies. The rGRA15-sensitized spleen cells from mice inoculated with the same antigen strongly promoted spleen cell proliferation and IFN-γ production. Immunization with rGRA15 significantly enhanced the survival rate of mice and dramatically decreased parasite burden in mice challenged with the Pru (type II) strain. These results suggested that rGRA15 triggered humoral and cellular immune responses to control infection. However, all of the immunized mice died when challenged with the GRA15-deficient Pru strain or the RH (type I) strain. These results suggest that GRA15 (II)-dependent immunity plays a crucial role in protection against challenge infection with the type II strain of T. gondii. This study is the first report to show GRA15 (II) as a recombinant vaccine antigen against Toxoplasma infection.

In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Clinical Comparison of Unilateral Biportal Endoscopic Laminectomy versus Microendoscopic Laminectomy for Single-Level Laminectomy: A Single-Center, Retrospective Analysis.

BACKGROUND: The purpose of this study was to compare clinical results of microendoscopic laminectomy (MEL) with those of unilateral biportal endoscopic laminectomy (UBEL) in patients with single-level lumbar spinal canal stenosis. METHODS: The subjects consisted of 181 patients who underwent MEL (139 cases) and UBEL (42 cases) who were followed up for at least 6 months. All patients had lumber canal stenosis for 1 level. Outcomes of the patients were assessed with the duration of surgery, the bone resection area in 3-dimensional computed tomography, the facet preservation rates in computed tomography axial imagery, Visual Analog Scale (VAS) for low back pain, the Oswestry Disability Index, and the EuroQol 5-Dimensions questionnaire. RESULTS: The bone resection area in 3-dimensional computed tomography was 1.5 for MEL versus 1.0 cm2 for UBEL (P < 0.05). The facet preservation rates on the advancing side and the opposite side were 78% versus 86% (advancing side: MEL vs. UBEL) and 85% versus 94% (opposite side) (P < 0.05). The VAS (low back pain) score, VAS (leg pain), Oswestry Disability Index, and EuroQol 5-Dimension questionnaire significantly dropped in both groups at the final period (P < 0.05), however, exhibiting no difference between the 2 groups at each period. MEL resulted in greater numbers of complications, including 5 cases of hematoma paralysis, 8 cases of dura injury, 2 cases of reoperation, as opposed to zero cases of hematoma paralysis and only 2 cases of dura injury resulting from UBEL. CONCLUSIONS: The UBEL method is a more useful technique than the MEL method as it requires a smaller bone resection area and produces fewer complications.

Use of an Internal Retractor for Percutaneous Full-Endoscopic Resection in Cervical Intervertebral Disc Herniation with a Posterior Approach.

STUDY DESIGN: A preliminary case series study. PURPOSE: For the safety of performing a posterior percutaneous full-endoscopic cervical discectomy. OVERVIEW OF LITERATURE: Because of the lack of space for inserting an outer sheath above the intervertebral disc in the spinal canal, grasping the herniated disc with forceps while retracting the nerves with the forceps itself was required. This procedure produces the risk of inadvertently injuring nerves because of inadequate visualization of the hernia and inadequate protection of the nerve. METHODS: Our new internal retractor can be inserted into the working channel of a percutaneous full-endoscope, enabling the insertion of a second tool. After partial foraminotomy, the internal retractor and forceps were manipulated to reliably retract the nerve root. Finally, the herniated disc was resected under an endoscopic view. RESULTS: All six cases had a good postoperative course, and postoperative neuropathy was not observed. CONCLUSIONS: This internal retractor allows for the secure resection herniated cervical intervertebral discs.

Methods and Early Clinical Results of Percutaneous Lumbar Interbody Fusion.

OBJECTIVE: Percutaneous lumbar interbody fusion (PELIF) is a procedure that includes the use of new devices, which allow minimally invasive diskectomy under the percutaneous full-endoscopic guidance and safe percutaneous insertion of a standard-sized cage. This procedure can be applied to severe disk degeneration, spondylolisthesis, and all lumbar intervertebral levels including the L5-S1 level. We report the methods and the clinical outcomes of this procedure. METHODS: Percutaneous diskectomy was performed with an outer sheath cutter and other devices. A cage was inserted with an L-shaped retract-slider. Hybrid facet screw fixation was performed for severe disk degeneration without spondylolisthesis. Conventional percutaneous pedicle screw fixation was performed for spondylolisthesis. The subjects consisted of 21 patients, who underwent PELIF and were followed up for 1 year or longer. RESULTS: No complications related to cage insertion were detected. The mean visual analogue scale scores were improved from 6.1 to 1.9 for lower back pain in severe disc degeneration cases without spondylolisthesis, and from 7.6 to 1.0 for lower extremity symptoms in spondylolisthesis cases. CONCLUSION: The clinical outcomes were favorable. PELIF was found to be a minimally invasive method that did not compromise safety and efficiency. PELIF is a possible therapeutic option that should be considered for not only spondylolisthesis at various intervertebral levels but also for severe disk degeneration because of its minimal invasiveness.

Postoperative Outcomes After Transsacral Epiduroscopic Laser Decompression in Japanese Patients: Denervation Therapy for Discogenic Low-Back Pain.

Background: Transsacral epiduroscopic laser decompression (SELD) is a very noninvasive surgery, so it is effective for elderly patients and athletes and is a new and minimally invasive therapeutic technique that may be useful in many patients with discogenic low-back pain (LBP) having high signal intensity zone (HIZ) in magnetic resonance imaging (MRI). We investigated the clinical outcomes of SELD in Japanese patients with discogenic LBP having HIZ as a first trial. Methods: The subjects consisted of 52 patients who underwent SELD and were followed up for at least 6 months. All patients with LBP with HIZ were operative using the SELD technique. Outcomes of the patients were assessed with visual analogue scale (VAS) for LBP, the Oswestry disability index (ODI), and the EuroQol 5 dimension (EQ-5D). Statistical analyses were carried out using a paired t-test. A p-value of <0.05 was considered significant. For statistical analysis, we used the SPSS software program. Results: At 12 months after the procedure, the average VAS score for LBP fell to 1.2 from 5.6 (p-value <0.05). The ODI score also dropped from the preoperative level of 22.3 to 8.8. The EQ-5D score also significantly increased from the preoperative level of 0.865 (SD 0.10) to 0.950 (SD 0.05). Eight cases of intraoperative cervical pain were observed as complications with no cases of hematomas, infections, and postoperative neurosis was observed. Conclusions: SELD provides a novel minimally invasive technique capable of performing multilevel intervertebral surgery. We believe that SELD is an effective method of treating discogenic LBP due to HIZs.

A New Percutaneous Endoscopic Instrument for Far-migrated Disk Herniation: A Retrospective Single-Institution Study.

OBJECTIVE: Compared with the traditional posterior approach, the transforaminal (TF) approach for percutaneous endoscopic lumbar diskectomy (PELD) in patients with disk herniation has a limited resection range that is problematic to treat far-migrated disk herniation. This study examined the usefulness of a newly developed percutaneous endoscopic instrument for far-migrated disk herniation. METHODS: This retrospective study included 22 patients with migrated disk herniation ≥ 10 mm who underwent transforaminal PELD using the new instrument between August 2014 and May 2017. The new instrument differs from conventional forceps because it has only one jaw that rotates in a plane perpendicular to the mouth opening. The migrated herniated disk is held between the instrument and bone surface, pulled out, and subsequently removed using conventional forceps. RESULTS: Herniated disks that had migrated up to 16 mm were removed successfully in all patients. CONCLUSIONS: The new instrument is a useful tool that can expand the resection range of the TF approach for PELD.

Step-by-Step Sublaminar Approach With a Newly-Designed Spinal Endoscope for Unilateral-Approach Bilateral Decompression in Spinal Stenosis.

OBJECTIVE: Spinal stenosis is increasingly common due to population aging. In elderly patients with lumbar central canal stenosis (LCCS), minimizing muscle damage and bone resection is particularly important. We performed a step-by-step operation with a newly designed spinal endoscope to obtain adequate decompression in patients with spinal stenosis. METHODS: From April 2015 to August 2016, 78 patients (48 males, 30 females) with LCCS (91 segments) underwent endoscopic decompression using a newly designed endoscope system. The inclusion criteria were: (1) neurogenic intermittent claudication with or without radiculopathy, (2) LCCS, and (3) having exhausted conservative treatment (>3 months). The exclusion criteria were: (1) >10° of instability, (2) spondylolisthesis grade II or greater according to the Meyerding criteria, (3) foraminal stenosis, (4) vascular intermittent claudication, (5) infection, and (6) stenosis combined with malignancy. We performed a step-by-step procedure using a newly designed endoscope system for unilateral-approach bilateral decompression. We used the same incision for 2-3 segments, only moving the skin. RESULTS: The mean follow-up was 2.3±1.3 years. Excellent or good results were found according to the MacNab criteria in 85.9% of cases (67 of 78). The visual analogue scale, Japanese Orthopedic Association score, and Oswestry Disability Index showed significant decreases at 1 month, persisting until the 2-year follow-up. Dural tear occurred in 4 cases (5.1%), and patch repair was performed under endoscopy. No patients experienced aggravated instability requiring surgery. CONCLUSION: We obtained good results with endoscopic decompression surgery using a newly designed instrument that minimized muscle and bone damage in elderly patients with spinal stenosis.

Percutaneous Endoscopic Cervical Discectomy: Surgical Approaches and Postoperative Imaging Changes.

STUDY DESIGN: Retrospective clinical study. PURPOSE: This study investigated the relationship between surgical approaches and surgical outcomes in patients undergoing percutaneous endoscopic cervical discectomy (PECD), including the reduction in intervertebral disc height and the incidence of Modic changes. OVERVIEW OF LITERATURE: The anterior approach involves partial invasion of the intervertebral disc, with a reported reduction in intervertebral disc height after PECD. METHODS: Forty-two patients with cervical disk hernia who underwent PECD and magnetic resonance imaging at least 3 months postoperatively were divided into four groups according to the hernia sites and the surgical approach used: unilateral hernia treated using the contralateral approach (group C, n=18), unilateral hernia treated using the ipsilateral approach (group I, n=15), midline hernia (group M, n=4), and broad and bilateral hernia (group B, n=5). Modic changes and intervertebral disc height were evaluated. RESULTS: The overall incidence of Modic changes was 52.4%: 72.2% in group C, 26.7% in group I, 25.0% in group M, and 80.0% in group B. The reduction in intervertebral disc height was 21.8% across all the patients: 24.5% in group C, 11.0% in group I, 22.8% in group M, and 23.9% in group B. CONCLUSIONS: The incidence of Modic changes and the reduction in intervertebral disc height were lower in the patients treated using the ipsilateral approach than in those treated using the contralateral approach. Traditionally, a contralateral approach has been used for PECD; however, the ipsilateral approach is more appropriate and is therefore recommended.

Spectral shift functions and Dirichlet-to-Neumann maps.

The spectral shift function of a pair of self-adjoint operators is expressed via an abstract operator-valued Titchmarsh-Weyl m-function. This general result is applied to different self-adjoint realizations of second-order elliptic partial differential operators on smooth domains with compact boundaries and Schrödinger operators with compactly supported potentials. In these applications the spectral shift function is determined in an explicit form with the help of (energy parameter dependent) Dirichlet-to-Neumann maps.

Full Percutaneous Lumbar Interbody Fusion: Technical Note.

Introduction: Although lumbar interbody fusion is effective for low back pain caused by severe disk degeneration, it is a highly invasive procedure. Less invasive procedures such as transforaminal lumbar interbody fusion (TLIF) and lumbar lateral interbody fusion have become available; however, there is still scope for improvement. We performed full percutaneous endoscopic lumbar interbody fusion (PELIF), a technique designed as a safe and less invasive percutaneous fusion. Method and Subjects: Our technique is indicated for patients with chronic low back pain in whom conservative treatment was not effective, thinning of the intervertebral disk was prominent, and temporary pain relief was achieved with a disk block. In the operation, percutaneous endoscopic diskectomy was performed with a 7.5-mm sheath inserted through a small incision, and a cage was inserted percutaneously using an L-shaped retractor. Instead of pedicle screw fixation, hybrid facet screw fixation was performed. Low back pain was improved, and bone union was confirmed on radiography. This technique was used in six patients, and no surgery-related complications occurred. Discussion: The L-shaped retractor used in this series can protect the exiting nerve by inserting it into the sheath, then removing the sheath and placing the rounded corner of the retractor on the lateral cranial side. This technique is safe with no other associated risks. Cages larger than the sheath can be inserted, and commercially available cages for TLIF are applicable. Hybrid facet screw fixation can overcome the problems associated with both conventional transfacet pedicle screw fixation and translaminar facet screw fixation by combining these two procedures. Conclusion: PELIF is an easy, safe, and fully percutaneous technique with very low invasiveness that uses an L-shaped retractor and hybrid facet screw fixation. This procedure can be a treatment option for patients with severe low back pain related to disk degeneration.

Rectangular Tubular Retractor for Microendoscopic Lumbar Decompression.

Background A cylindrical working tube with a diameter of 16 mm has been used for endoscopic posterior lumbar spinal surgery. However, intraoperative muscle resection is significant when using the current conventional tubular retractor. Objective To describe a novel tubular retractor for microendoscopic surgery and to analyze the outcomes of lumbar decompressive laminotomy using this retractor. Materials and Methods We devised a novel tubular retractor by changing the medial and lateral sides of the conventional 16-mm cylindrical tubular retractor to planes with a mediolateral dimension of 10 mm (rectangular tubular retractor hereafter). The amount of muscle resection, osteotomy angle on the approach side, and operating time were compared between 25 intervertebral levels treated by bilateral decompression through a unilateral approach using the rectangular tubular retractor and 31 intervertebral levels treated with the same surgery using a 16-mm cylindrical tubular retractor. Results Due to the short mediolateral dimension, muscle resection decreased by 86%. The rectangular tubular retractor also decreased early postoperative wound pain. Because the craniocaudal dimension of the tubular retractor was maintained, surgical difficulty did not increase, resulting in only a slight increase in operating time. The facet joint on the approach side could be sufficiently preserved. Conclusions The rectangular tubular retractor reduced surgical invasiveness without increasing surgical difficulty.

Area of Ostectomy in Posterior Percutaneous Endoscopic Cervical Foraminotomy: Images and Mid-term Outcomes.

STUDY DESIGN: Retrospective. PURPOSE: To analyze whether the cross-sectional area of the intervertebral foramen at the outermost edge of the resection site is associated with postoperative outcomes and whether our fluoroscopic method for determining the resection area is appropriate. OVERVIEW OF LITERATURE: There is no consensus on the criteria for determining an optimal resection area to obtain sufficient decompression while maintaining intervertebral stability in posterior percutaneous endoscopic cervical foraminotomy. Previous reports have recommended a facet resection rate (FRR) of ≤50%. Intervertebral foramen stenosis often extends to the exit zone. The cross-sectional area of the intervertebral foramen is occasionally small at the outermost edge of the resection site. No report has analyzed whether these aspects are associated with postoperative outcomes. METHODS: Lateral margins of the resection area were set at lateral borders of the vertebral body on frontal fluoroscopic view. Because the percutaneous endoscope has a small diameter, surrounding structures can easily be identified using frontal view fluoroscopy to determine the resection area. FRRs were calculated from postoperative computed tomography images. The smallest cross-sectional area of the intervertebral foramen around the lateral edge of the resection area (SALE) was measured and compared wit clinical outcomes. RESULTS: The mean FRR was 41.7% at C5-C6 and 48.9% at C6-C7. SALE was not correlated with clinical outcomes. CONCLUSIONS: Residual stenosis in the lateral portion of the intervertebral foramen is weakly associated with postoperative outcomes. Our process achieved adequate FRRs and favorable postoperative outcomes, suggesting that our criteria for determining the resection area are appropriate.

A New Angled Chisel for Microendoscopic Decompressive Laminotomy.

STUDY DESIGN: A retrospective comparative series study. OBJECTIVE: The aim of the study was to describe a new angled chisel (NAC) that facilitates the osteotomy in microendoscopic decompressive laminotomy (MEDL), and to analyze the clinical and radiologic outcomes using the tool. SUMMARY OF BACKGROUND DATA: MEDL for lumbar spinal stenosis is a minimally invasive surgery. The paraspinous unilateral approach for bilateral decompression can preserve the posterior structure better than other methods. However, the resection of the medial facet on the approach side is technically difficult because the working space is limited and the retractor is difficult to place properly. Because of these limitations, either the inferior articular process on the approach side tends to be resected excessively, which can lead to facet fracture and instability, or the superior articular process tends to be resected insufficiently, which can result in residual symptoms. The ideal decompression of the medial facet consists of sufficient resection to the deep portion, especially lateral recess of the spinal canal, and adequate facet preservation. Special curved devices to obtain optimal resection have been developed, but these devices have not effectively improved the osteotomy. We developed an NAC to allow an osteotomy at the desired angle. MATERIALS AND METHODS: Forty patients underwent MEDL with the use of NAC (NAC group) and 40 patients underwent the same procedure without the NAC (control group). The osteotomy angle of the medial facet on the approach side and Visual Analogue Scale score were analyzed. RESULTS: The average osteotomy angle of the medial facet was significantly smaller in the NAC group. The radiologic and clinical results were significantly better in the NAC group. CONCLUSIONS: The NAC was a useful tool that sharpens the osteotomy angle of the medial facet and thereby improved the clinical course.

Hydrostatic pressure induces apoptosis of chondrocytes cultured in alginate beads.

The purpose of this study was to investigate the influence of hydrostatic pressure (HP) on apoptosis and expression of heat-shock protein 70 (HSP70) in chondrocytes cultured in alginate beads. Chondrocytes were isolated from the articular cartilage of rabbit joints and seeded in alginate beads. The beads in Group A were cultured for less than 24 h after being embedded with the chondrocytes, while those in Group B were cultured for 2 weeks. Both groups were exposed to HP of 10 or 50 MPa for 12 or 24 h. The beads in Groups A and B that were not exposed to HP were regarded as controls. Apoptotic cells induced by exposure to HP were quantified using the TUNEL method. Immunohistochemical analysis for HSP70 and in situ TUNEL analysis were also performed. Apoptotic chondrocytes were not observed in the control cells under atmospheric pressure, whereas apoptosis was observed in the beads in Group A, and the number of apoptotic cells increased as the duration and magnitude of HP increased. On the other hand, we observed no significant population of apoptotic cells in the beads in Group B. Chondrocytes expressing HSP70 were not TUNEL positive in the histological analysis. Excessively strong HP could evoke apoptosis when the extracellular matrix did not accumulate around the chondrocytes. HSP70 expression was related to occurrence of apoptosis that resulted from HP. These findings suggest a mechanism for the pathogenesis of cartilage degeneration in osteoarthritis.

Urokinase-Treated Antithrombogenic Drains and Optimized Drain Placement in Endoscopic Lumbar Decompressive Surgery.

Background Spinal epidural hematoma (SEH) frequently occurs after microendoscopic decompressive laminotomy (MEDL), and a drain may not be functioning sufficiently. Objective To reduce the incidence of SEH after MEDL. Methods A urokinase-treated antithrombogenic drain, which is available only with a large diameter, was reduced in diameter and used after MEDL. Magnetic resonance imaging (MRI) and computed tomography (CT) were performed 36 to 48 hours after surgery. The size of the SEH was measured by MRI, and the location of the drain tip was assessed by CT scan. After imaging, the drain was removed. Results Use of the urokinase-treated antithrombogenic drain reduced the incidence of SEH. However, the drain was not adequately placed in many cases, limiting the effect of the drainage. When the urokinase-treated antithrombogenic drain was placed contralaterally to the approach side using an unsheathed endoscope, the incidence of SEH was further reduced. Conclusions The urokinase-treated antithrombogenic drain prevented thrombus-related drain obstruction. In addition, unsheathed endoscopic contralateral placement of the drain was effective for SEH prevention.