Publications by authors named "Hong-Liang Zhao"

38 Publications

Full-Endoscopic Oblique Lateral Lumbar Interbody Fusion: A Technical Note With 1-Year Follow-Up.

Int J Spine Surg 2021 Jun 7;15(3):504-513. Epub 2021 May 7.

Desert Institute for Spine Care, Phoenix, Arizona; Executive Director International Intradiscal Therapy Society, Phoenix, Arizona.

Background: Oblique lateral lumbar interbody fusion (OLLIF) is a minimally invasive lumbar interbody fusion procedure using a bullet-shaped polyetheretherketone (PEEK) nonexpandable fusion cage modified to diminish risk to the exiting nerve root during posterolateral implantation through the Kambin safe zone under fluoroscopic guidance. The objective of this study was to present feasibility of this procedure and 1-year clinical outcome data.

Methods: The authors present a prospective cohort study of 20 patients who underwent fluoroscopy-guided and full-endoscopic OLLIF in 22 segments allowing protection of the exiting nerve root from January 2018 to March 2019. The foraminoplasty, discectomy, endplate preparation, placement of bone graft and insertion of the fusion cage was done under continuous full-endoscopic visualization. The OLLIF fusion was backed up with bilateral percutaneous posterior supplemental pedicle screw fixation. Primary clinical outcome measures were the visual analog scale (VAS) of low back and leg pain, and Oswestry disability index (ODI) at 1 week, 3 months, 6 months, and 1 year after the operation. At final follow-up, the Macnab score was also evaluated. Secondary outcome measures were computed tomography (CT) assessment fusion using the Mannion classification of spinal fusion and adverse events related to the device as well as magnetic resonance imaging (MRI) assessment of nerve root decompression.

Results: All patients had significant relief of low back pain and leg pain, by VAS and ODI scores that improved significantly ( < .01). There were no complications. Postoperative lumbar MRI of all patients showed sufficient direct nerve decompression. At 1-year follow-up, excellent Macnab outcomes were obtained 13 patients, good in six, and fair in one. Impaired sensation and muscle strength of the involved nerve root significantly recovered in all but 2 patients ( < .05). According to the Mannion CT-based classification of spinal fusion, CT showed complete interbody fusion achieved in all 22 segments.

Conclusions: Full-endoscopic OLLIF is a safe, effective, minimally invasive, economical, practical, and widely applicable minimally invasive interbody fusion technique in the lumbar spine.

Level Of Evidence: 3.
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http://dx.doi.org/10.14444/8072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176836PMC
June 2021

Ultrasonic Osteotome Assisted Full-Endoscopic en Block Resection of Thoracic Ossified Ligamentum Flavum: Technical Note and 2 Years Follow-up.

Pain Physician 2021 Mar;24(2):E239-E248

Department of Orthopedic Surgery, the Fourth Medical Center of Chinese PLA General Hospital, Beijing, China.

Background: Conventional open laminectomy is considered to be the standard procedure for the treatment of thoracic ossified ligamentum flavum, but multi-segment thoracic laminectomy extensively removes the facet joints and ligamentous tissue, destroying the thoracic spine biomechanics and stability, may lead to delayed thoracic spine kyphosis deformities, which in turn can lead to potential neurological deterioration and local intractable pain.

Objective: To introduce the technical notes and clinical outcome of ultrasonic osteotome assisted full-endoscopic en block resection of thoracic ossified ligamentum flavum.

Study Design: A prospective cohort study.

Setting: Hospital and outpatient surgery center.

Methods: From January 2017 to March 2018, 15 patients with 1 - 2 segment thoracic ossified ligamentum flavum were treated with ultrasonic osteotome assisted full-endoscopic en block resection of thoracic ossified ligamentum flavum under local anesthesia. The magnetic resonance imaging and computed tomography of the thoracic spine was reexamined after the operation to evaluate the completeness of ossified ligamentum flavum resection and spinal cord decompression. The patients were followed up on the visual analog scale of back pain and radicular pain, Nurick score and mJOA score of neurological function, and Oswestry Disability Index at 1 week, 3 months, 6 months, one year, and 2 years after operation.

Results: All operations of 17 segments thoracic ossified ligamentum flavum in 15 patients were successfully completed without intraoperative conversion to open surgery. There were no intraoperative spinal cord injuries, dura tears, postoperative cerebrospinal fluid leakage, postoperative infections, and postoperative spinal cord injury aggravated symptoms. Postoperative thoracic spine magnetic resonance imaging and computed tomography examinations of all patients showed that the spinal cord was fully decompressed without any residual pressure. Back pain and radicular pain were relieved significantly, and spinal cord function (Nurick, mJOA, and Oswestry Disability Index scores) was obviously restored. The mJOA recovery rate at the 2-year follow-up was 78.3% in average.

Limitations: This is an observational cohort study with relative small sample and short-term follow-up.

Conclusions: Ultrasonic assisted full-endoscopic en block resection of ossified ligamentum flavum is a safe and effective minimally invasive spine surgery for thoracic myelography caused by thoracic ossified ligamentum flavum.
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March 2021

Complete plastome sequence of G. H. Huang & G. Hao, an endemic species in Hainan.

Mitochondrial DNA B Resour 2020 Jun 22;5(3):2531-2532. Epub 2020 Jun 22.

School of Life and Pharmaceutical Sciences, Hainan University, Haikou, China.

G. H. Huang & G. Hao is a small shrubs of Primulaceae. It is only distributed in Hainan provinces of China. It is a plant medicinal value. There is no study on the genome of so far. Here, we report and characterize the complete plastid genome sequence of in an order to provide genomic resources useful for promoting its conservation. The complete chloroplast genome of is 160,176 bp in length with a typical quadripartite structure, consisting of a large single-copy region (LSC, 89,710 bp), a single-copy region (SSC, 18,357 bp), and a pair of inverted repeats (IRs, 26,054 bp). There are 133 genes annotated, including 83 unique protein-coding genes, eight unique ribosomal RNA genes, and 37 transfer RNA genes. The overall G/C content in the plastome of is 36.0%. The complete plastome sequence of will provide a useful resource for the conservation genetics of this species as well as for phylogenetic studies in Apocynaceae.
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http://dx.doi.org/10.1080/23802359.2020.1778577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782065PMC
June 2020

Protective effects of HBOC on pulmonary vascular leakage after haemorrhagic shock and the underlying mechanisms.

Artif Cells Nanomed Biotechnol 2020 Dec;48(1):1272-1281

State Key Laboratory of Trauma, Burns and Combined Injury, Second Department of Research Institute of Surgery, Daping Hospital, Army Medical University, Chongqing, P.R. China.

Volume resuscitation is an important early treatment for haemorrhagic shock. Haemoglobin-based oxygen carrier (HBOC) can expand the volume and provide oxygen for tissues. Vascular leakage is common complication in the process of haemorrhagic shock and resuscitation. The aim of this study was to observe the effects of HBOC (a bovine-derived, cross-linked tetramer haemoglobin oxygen-carrying solution, 0.5 g/L) on vascular leakage in rats after haemorrhagic shock. A haemorrhagic shock rat model and hypoxic vascular endothelial cells (VECs) were used. The role of intercellular junctions and endothelial glycocalyx in the protective effects of HBOC and the relationship with mitochondrial function were analysed. After haemorrhagic shock, the pulmonary vascular permeability to FITC-BSA, Evans Blue was increased, endothelial glycocalyx was destroyed and the expression of intercellular junction proteins was decreased. After haemorrhagic shock, a small volume of HBOC solution (6 ml/kg) protected pulmonary vascular permeability, increased structural thickness of endothelial glycocalyx, the levels of its components and increased expression levels of the intercellular junction proteins ZO-1, VE-cadherin and occludin. Moreover, HBOC significantly increased oxygen delivery and consumption in rats, improved VEC mitochondrial function and structure. In conclusion, HBOC mitigates endothelial leakage by protecting endothelial glycocalyx and intercellular junctions through improving mitochondrial function and tissue oxygen delivery.
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http://dx.doi.org/10.1080/21691401.2020.1835937DOI Listing
December 2020

Safety and Efficacy of the Metabolic Profiling of the BIMRT Utilizing 18F FDG PET-CT.

Technol Cancer Res Treat 2020 Jan-Dec;19:1533033820960723

Department of Obstetrics and Gynecology, Taizhou Hospital of Zhejiang Province, Taizhou Enze Medical Center (Group), Linhai, Zhejiang Province, China.

Aim: This study aims to evaluate the safety and efficacy of fluorodeoxyglucose positron emission tomography/computed tomography (F-FDG-PET/CT) guided intensity-modulated radiation therapy (IMRT) for patients with peritoneal metastases.

Patients And Methods: A total of 55 patients with peritoneal metastases were treated with 18F-FDG-PET/CT-guided IMRT (BIMRT) from January 2012 to January 2019. They were prescribed with a fraction of the median dose of 2 Gy to a total dose of 50.4 Gy. The multivariate analysis was used the Cox proportional hazard model and the Kaplan-Meier plot was used to perform local control rate (LCR), progression-free survival (PFS), and overall survival (OS) analysis.

Results: The 1-year, 2-year, and 3-year LCR were 72.7%, 36.4%, and 9.1%, respectively; the 1-year, 2-year, and 3-year PFS were 69.1%, 30.9%, and 7.3%, respectively, and the median PFS time was 18 months. The 1-year, 3-year and 5-year OS were 70.9%, 28.7%, and 4.2%, respectively. Based on the multivariate analysis using the Cox proportional hazard model, the Karnofsky performance status (KPS) score and radiotherapy joint chemotherapy (RJC) method were independent prognostic-related indicators (P < 0.0001).

Conclusion: BIMRT may be a safe and effective treatment for patients with peritoneal metastases, especially for patients who cannot undergo surgery. In addition, the results indicated that the patient's KPS score and RJC method were independent prognostic-related indicators for patients survival time.
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http://dx.doi.org/10.1177/1533033820960723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534080PMC
September 2020

A Pilot Study of Full-Endoscopic Annulus Fibrosus Suture Following Lumbar Discectomy: Technique Notes and One-Year Follow-Up.

Pain Physician 2020 09;23(5):E497-E506

The First Affiliated Hospital of Chinese PLA's General Hospital, Beijing, China.

Background: An annulus fissure or defect will inevitably be left on the posterior annulus fibrosus after almost all kinds of lumbar discectomy, which may lead to unsatisfying postoperative pain relief and recurrence of the disc herniation.

Objective: The objective of this research is to introduce the technique of full-endoscopic annulus fibrosus suture following lumbar discectomy through the transforaminal or interlaminar approach, and to analyze the clinical outcome of full-endoscopic lumbar discectomy and annulus fibrosus suture.

Study Design: This study used a prospective cohort design.

Setting: The research was conducted in a hospital and outpatient surgery center.

Methods: A total of 50 patients with noncontained lumbar disc herniation treated with full-endoscopic lumbar discectomy and annulus fibrosus suture were treated in our department between January 2018 and November 2018. Full-endoscopic single-stitch suture via the transforaminal approach (Group T) or double-stitch suture via the interlaminar approach (Group I) was selected according to the level of lesion. Lumbar magnetic resonance imaging (MRI) was reexamined on the second day and 3 months after operation to evaluate the completeness of the discectomy and the adequacy of nerve decompression. Patients were followed up on the second day, 3 months, 6 months, and one year after operation to evaluate the relief of low back pain and leg pain, using a visual analog scale (VAS, 100-point scale). At 3 months, 6 months, and one year after operation, the patients were followed up for recovery of lumbar spine function, using the Oswestry Disability Index (ODI). At the one-year follow-up, the MacNab score was used to evaluate the clinical outcome, and the recovery of nerve root function (sensation, muscle strength, and reflex) was recorded.

Results: All operations were successfully completed, including 27 cases in Group T and 23 cases in Group I. There were no surgical complications and no recurrence of lumbar disc herniation. Lumbar MRI reexaminations of all patients showed that the herniated disc was completely removed and the nerves were fully decompressed. Postoperative low back pain and leg pain were significantly relieved, and the ODI score was significantly improved (P < .01) in both groups. At the one-year follow-up, the excellent and good rates as measured by the MacNab score were 92.6% in Group T and 91.3% in Group I with no significant difference between the 2 groups (P > .05). The impaired sensation and muscle strength in the low extremities of evolved nerve root of the 2 groups of patients recovered significantly at the one-year follow-up (P < .01), but the tendon reflex did not recover significantly (P > .05).

Limitations: This is an observational cohort study with relatively small sample sizes and short-term follow-up.

Conclusions: Full-endoscopic lumbar discectomy and annulus fibrosus suture through either the transforaminal or interlaminar approach are safe and effective minimally invasive spinal surgery techniques that can reduce the recurrence rate of lumbar disc herniation after full-endoscopic lumbar discectomy.
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September 2020

18β-Glycyrrhetinic Acid Improves Cardiac Diastolic Function by Attenuating Intracellular Calcium Overload.

Curr Med Sci 2020 Aug 29;40(4):654-661. Epub 2020 Aug 29.

Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.

Ranolazine, a late sodium current inhibitor, has been demonstrated to be effective on heart failure. 18β-glycyrrhetinic acid (18β-GA) has the similar inhibitory effect on late sodium currents. However, its effect on diastolic function is still unknown. This study aimed to determine whether 18β-GA can improve the diastolic function and to explore the underlying mechanisms. Eighty male Sprague Dawley (SD) rats of Langendorff model were randomly divided into the following groups: group A, normal cardiac perfusion group; group B, ischemia-reperfusion group; group C, ischemia-reperfusion with anemoniasulcata toxin II (ATX-II); group D, ranolazine group; and group E, 18β-GA group with four different concentrations. Furthermore, a pressure-overloaded rat model induced by trans-aortic constriction (TAC) was established. Echocardiography and hemodynamics were used to evaluate diastolic function at 14th day after TAC. Changes of free intracellular calcium (Ca) concentration was indirectly detected by laser scanning confocal microscope to confirm the inhibition of late sodium currents. With the intervention of ATX-II on ischemia reperfusion injury group, 5 µmol/L ranolazine, and 5, 10, 20, 40 µmol/L 18β-GA could improve ATX-II-induced cardiac diastolic dysfunction. 630 mg/kg glycyrrhizin tablets could improve cardiac diastolic function in the pressure-overloaded rats. 18β-GA and ranolazine had similar effects on reducing the free calcium in cardiomyocytes. The study demonstrates that 18β-GA and glycyrrhizin could improve diastolic dysfunction induced by ischemia-reperfusion injury in Langendorff-perfused rat hearts and pressure-overloaded rats. The mechanism may be attributed to the inhibition of enhanced late sodium currents.
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http://dx.doi.org/10.1007/s11596-020-2232-yDOI Listing
August 2020

[Analysis of the technical key points and clinical effect of full-endoscopic lumbar annulus fibrosus suture].

Zhongguo Gu Shang 2020 Jun;33(6):498-504

Department of Orthopaedics, the Fourth Medical Center of Chinese PLA General Hospital, Beijing 100048, China.

Objective: To introduce the technical key points of lumbar annulus fibrosus suture under full-endoscope and analyze the clinical efficacy of full-endoscopic lumbar discectomy and annulus fibrosus suture.

Methods: A total of 50 patients with non contained lumbar disc herniation treated with full-endoscopic lumbar discectomy and annulus fibrosus suture in our department between January 2018 and November 2018 were included. Full-endoscopic single-stitch suture through transforaminal approach or double-stitch suture through interlaminar approach was selected according to lesion level. The lumbar MRI and CT were reexamined on the second day and 3 months after surgery to evaluate the completeness of the discectomy and the adequacy of nerve decompression respectively. The patients were followed up on the second day, 3 months, 6 months, and 1 year after surgery for pain relief using visual analogue scale (VAS, 100 -point scale). The patients were followed up at 3 months, 6 months, and 1 year postoperatively for the recovery of lumbar spine function using Oswestry Disability Index(ODI). At the 1-year follow-up, the Macnab standard of lumbar spine function was evaluated, and the recovery of nerve root function (sensory, muscular and reflex) was recorded.

Results: All operations were successfully completed, of which 27 patients were treated with transforaminal approach(including 8 cases of L and 19 cases of L), and 23 patients(including 11 cases of L and 12 cases of LS) with interlaminar approach. The average operation time was 43.2 minutes. There were no surgical complications and no recurrence of lumbar disc herniation. Postoperative lumbar MRI and CT examinations of all patients showed that the herniated disc was completely removed and the nerveswere fully decompressed. All patients had significant relief of low back pain and lower extremity radiation pain, and the ODI score improved significantly(<0.01). At 1 year postoperative follow up, 17 patients got an excellent result, 29 good and 4 fair according to Macnab evaluation system. On the first year after surgery, the sense of damaged nerve roots and muscle strength were significantly restored (<0.01), but tendon reflexes were not significantly restored (>0.05).

Conclusion: Full-endoscopic lumbar discectomy and annulus fibrosus suture are safe and effective techniques for minimally invasive spinal surgery, which can reduce the recurrence rate of lumbar disc herniation after full endoscopic lumbar discectomy.
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http://dx.doi.org/10.12200/j.issn.1003-0034.2020.06.002DOI Listing
June 2020

Percutaneous Isthmus Foraminoplasty and Full-Endoscopic Lumbar Discectomy for Very Highly Upmigrated Lumbar Disc Herniation: Technique Notes and 2 Years Follow-Up.

World Neurosurg 2020 09 9;141:e9-e17. Epub 2020 Apr 9.

Department of Orthopedics, the Fourth Medical Center of Chinese PLA General Hospital, Beijing, China.

Objective: To report the technical notes and clinical outcome of percutaneous isthmus foraminoplasty and full-endoscopic lumbar discectomy (PIF-FELD) for the treatment of very highly upmigrated lumbar disc herniation (VHUM-LDH).

Patients And Methods: From January 2014 to December 2017, 16 patients with VHUM-LDH underwent PIF-FELD surgery. A 10-mm-diameter semi-open foraminoplasty was performed on the dorsal side of the upper intervertebral foramen with lateral isthmus resection by a safe trephine system, in which the facet joint was not injured. Full-endoscopic transforaminal fragmentectomy in the spinal canal and discectomy in the intervertebral space were performed simultaneously. Magnetic resonance imaging of the lumbar spine was reexamined on the second day and 3 months after the operation to evaluate the completeness of the disc fragmentectomy and nerve decompression. The patients were followed up on the visual analog scale (VAS) of lumbar pain and leg pain and Oswestry Disability Index on the second day, 3 months, 6 months, 1 year, and 2 years after operation. The MacNab score and nerve root function recovery were evaluated at 2 years postoperative follow-up.

Results: All operations were successfully completed without any change in the surgical method. During the operation, 5 patients were found to have a single sequestered fragment, and the remaining 11 patients had multiple sequestered fragments. There were no complications (e.g., dural tear and nerve injury) during the operation, and no complications (e.g., infection, cerebrospinal fluid leakage, exacerbation of nerve root function injury, and recurrence of intervertebral disc herniation) occurred. Postoperative reexamination of lumbar magnetic resonance imaging in all patients showed complete disc fragmentectomy and adequate nerve decompression. The postoperative low back pain VAS and leg pain VAS scores and Oswestry Disability Index on the second day, 3 months, 6 months, 1 year, and 2 years after operation were significantly improved compared with preoperatively (P < 0.01). At 2 years follow-up, the sensation and muscular strength of the affected nerve root innervation area were significantly restored (P < 0.05), but tendon reflex function was not significantly restored (P > 0.05). At 2 years follow-up, MacNab scores included 6 excellent, 9 good, and 1 fair.

Conclusions: PIF-FELD is a safe and effective minimally invasive spine surgery technique for VHUM-LDH.
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http://dx.doi.org/10.1016/j.wneu.2020.03.141DOI Listing
September 2020

Role of resveratrol in protecting vasodilatation function in septic shock rats and its mechanism.

J Trauma Acute Care Surg 2019 12;87(6):1336-1345

From the State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Research Institute of Surgery, Daping Hospital, Army Medical University, Chongqing, China.

Background: Vascular dysfunction is a major cause of sepsis-induced multiple-organ dysfunction. Resveratrol is a polyphenol compound with extensive pharmacological effects including anti-inflammation. The aim of this study was to determine the role and mechanism of resveratrol in protecting vascular function following sepsis.

Methods: The cecal ligation and puncture method was used to establish a septic shock rat model. Resveratrol (5 mg/kg and 10 mg/kg) was administered intravenously immediately and at 12 hours after cecal ligation and puncture, respectively. The effects of resveratrol on vasodilatation function, blood flow velocity, hemodynamics, and vital organ function and its relationship to Rac-1 and HIF-1α were observed.

Results: Vascular relaxation reactivity and blood flow velocity were significantly decreased after septic shock, both were significantly improved by resveratrol 5 mg/kg and 10 mg/kg, and the effect of 10 mg/kg was greater. The relaxation reactivity of the superior mesenteric artery to acetylcholine (Ach) was increased by 43.2%. The blood flow velocity of mesenteric arterioles and venules was increased by 47.1% and 51%, respectively, after resveratrol (10 mg/kg) administration compared with the septic shock group. The hemodynamics and both liver and kidney blood flow were significantly decreased after septic shock, which were significantly improved them by resveratrol, which enhanced the vascular relaxation reactivity in septic shock rats. The 72-hour survival rate of septic shock rats in the resveratrol group (62.5%) was significantly higher than that in the septic shock group (6.3%). Resveratrol significantly upregulated the expression of endothelial nitric oxide synthase (eNOS) and downregulated the expression of inducible NOS, Rac-1, and HIF-1α. Inhibitors of Rac-1 and HIF-1α significantly improved the expression of eNOS, and inhibition of eNOS (L-NAME, 5 mg/kg) antagonized the resveratrol-induced improvement in vascular relaxation reactivity and survival.

Conclusion: Resveratrol was beneficial for vasodilatation function in rats with septic shock, which is the major contribution to resveratrol improving hemodynamics and organ perfusion. The mechanism involved resveratrol upregulating the expression of eNOS by inhibiting Rac-1 and HIF-1α.
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http://dx.doi.org/10.1097/TA.0000000000002466DOI Listing
December 2019

Non-concomitant cortical structural and functional alterations in sensorimotor areas following incomplete spinal cord injury.

Neural Regen Res 2017 Dec;12(12):2059-2066

Department of Radiology, Beijing Tsinghua Changgung Hospital, Beijing, China.

Brain plasticity, including anatomical changes and functional reorganization, is the physiological basis of functional recovery after spinal cord injury (SCI). The correlation between brain anatomical changes and functional reorganization after SCI is unclear. This study aimed to explore whether alterations of cortical structure and network function are concomitant in sensorimotor areas after incomplete SCI. Eighteen patients with incomplete SCI (mean age 40.94 ± 14.10 years old; male:female, 7:11) and 18 healthy subjects (37.33 ± 11.79 years old; male:female, 7:11) were studied by resting state functional magnetic resonance imaging. Gray matter volume (GMV) and functional connectivity were used to evaluate cortical structure and network function, respectively. There was no significant alteration of GMV in sensorimotor areas in patients with incomplete SCI compared with healthy subjects. Intra-hemispheric functional connectivity between left primary somatosensory cortex (BA1) and left primary motor cortex (BA4), and left BA1 and left somatosensory association cortex (BA5) was decreased, as well as inter-hemispheric functional connectivity between left BA1 and right BA4, left BA1 and right BA5, and left BA4 and right BA5 in patients with SCI. Functional connectivity between both BA4 areas was also decreased. The decreased functional connectivity between the left BA1 and the right BA4 positively correlated with American Spinal Injury Association sensory score in SCI patients. The results indicate that alterations of cortical anatomical structure and network functional connectivity in sensorimotor areas were non-concomitant in patients with incomplete SCI, indicating the network functional changes in sensorimotor areas may not be dependent on anatomic structure. The strength of functional connectivity within sensorimotor areas could serve as a potential imaging biomarker for assessment and prediction of sensory function in patients with incomplete SCI. This trial was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR-ROC-17013566).
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http://dx.doi.org/10.4103/1673-5374.221165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784355PMC
December 2017

Modified Percutaneous Lumbar Foraminoplasty and Percutaneous Endoscopic Lumbar Discectomy: Instrument Design, Technique Notes, and 5 Years Follow-up.

Pain Physician 2017 Jan-Feb;20(1):E85-E98

The First Affiliated Hospital of Chinese PLA's General Hospital, Beijing, China.

Background: Conventional percutaneous endoscopic lumbar discectomy (PELD) with an "inside-outside" technique has 4.3% - 10.3% surgical failure rate, especially in central herniated discs (HDs), migrated HDs, and axillary type HDs. PELD with foraminoplasty has been used for complex HDs. Percutaneous lumbar foraminoplasty (PLF), which is performed with a trephine or bone reamer introduced over a guidewire without a protective working cannula in the original Tessys technique, can quickly cut the hypertrophied bony structure under fluoroscopic guidance, and risk injury to the exiting and traversing nerve roots.

Study Design: A prospective cohort study.

Setting: Hospital and outpatient surgical center.

Objective: To evaluate the outcome and safety of modified PLF-PELD with a specially designed instrument for complex uncontained lumbar HDs.

Method: From April of 2007 to April of 2009, 148 patients with uncontained lumbar HDs were treated with modified PLF-PELD. Magnetic resonance imaging (MRI) checkup was performed the next morning after the operation. Outcomes of symptoms were evaluated by follow-up interviews at 3 months, 6 months, one year, and 5 years after surgery. Low back pain and leg pain were measured by visual analog scale (VAS) score (1 - 100). Functional outcomes were assessed by using the Oswestry Disability Index (ODI) and modified MacNab criteria.

Results: Follow-up data were obtained from 134 cases, including 14 cases on L3-4, 78 cases on L4-5, and 42 cases on L5-S1. One hundred-eight cases were prolapse type, while 26 cases were sequestration type. Pre-operative symptoms and deficits included nerve root dermatome hypoesthesia in 98 patients (73%), nerve root myotome muscle weakness in 32 patients (23%), and weakening or disappearance of tendon reflex in 43 patients (32%). No case required conversion to an open procedure during the surgery. Low back pain and leg pain were significantly relieved immediately after surgery in all patients. MRI examination showed adequate removal of HD in all patients. VAS scores and ODI values were significantly lower at all time points after surgery than before surgery. The percentage of pain relief in leg pain was significantly higher than that in low back pain (P < 0.01). But there was no significant correlation between duration of the preoperative symptoms and the percentage of pain relief. MacNab scores at 5 years after surgery were obtained from 134 patients. Seventy-five cases were rated "excellent"; 49 were rated "good," Five patients experienced heavier low back pain, thus being classified as "fair." Five cases with recurrence were rated "poor." Preoperative and postoperative (5 years follow-up) related nerve root function status was compared. Sensation and muscle strength recovered significantly (P < 0.01), while tendon reflex was not changed (P = 0.782). No patients had infections. Five patients were complicated with dysesthesia in distribution of the exiting nerve that was all operated at L5-S1. Complaints were reduced one week after treatment with medium frequency pulse electrotherapy. Five cases required a revision surgery after recurrence.

Limitations: This is an observational clinical case series study without comparison.

Conclusion: Modified PLF-PELD with a specially designed instrument is a less invasive, effective and safe surgery for complex uncontained lumbar DH.Key words: Lumbar disc herniation, minimally invasive treatment, foraminoplasty, percutaneous endoscopic lumbar discectomy.
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July 2017

Percutaneous lumbar foraminoplasty and percutaneous endoscopic lumbar decompression for lateral recess stenosis through transforaminal approach: Technique notes and 2 years follow-up.

Clin Neurol Neurosurg 2016 Apr 10;143:90-4. Epub 2016 Feb 10.

The Department of Orthopedic Surgery, The First Affiliated Hospital of Chinese PLA's General Hospital, People's Republic of China.

Objective: To evaluate the outcome and safety of percutaneous lumbar foraminoplasty (PLF) and percutaneous endoscopic lumbar decompression (PELD) with specially designed instrument for lumbar lateral recess stenosis with/without herniated discs (HDs).

Method: From August of 2011 to August of 2013, 96 patients met the inclusion criteria were treated with PLF-PELD and 85 cases were followed up to 2 years postoperatively. MRI or CT checkup performed in the next morning after operation. Outcomes of symptoms were evaluated by follow-up interviews at 3 months, 6 months, 1 year and 2 years after surgery. Low back pain and leg pain were measured by Visual Analog Scale (VAS) score (1-100). Functional outcomes were assessed by using Oswestry Disability Index (ODI) and modified MacNab criteria.

Result: Two years follow-up data were obtained from 85 cases, including 14 cases on unilateral L3-4, 1 case on bilateral L3-4, 49 cases on unilateral L4-5, 3 cases on bilateral L4-5, 12 cases on unilateral L5S1, 1 case on bilateral L5S1, 3 cases on unilateral L3-5 and 2 cases on unilateral L4-S1. So totally 95 lumbar lateral recesses were decompressed. Patients ranged in age from 46-78 years (mean age, 56.7 years), including 36 males and 49 females. 56 cases combined with HDs. Low back pain and leg pain were significantly relieved after surgery in all patients. 3 patients were complicated with dysesthesia in distribution of exiting nerve that was all operated at L5S1. Postoperative MRI/CT examination showed adequate decompression of lateral recess and removal of combined HDs in all patients. No patient had postoperative infection, dysfunctional nerve root injury or iatrogenic segmental instability. 2 cases experienced recurrence of combined HDs (2.4%), but could not undertake further revision surgery because of infirm condition. All the 85 cases were analyzed with complete follow-up data. VAS scores and ODI values were significantly lower in all time-points after surgery than before surgery. MacNab scores at 2 years after surgery were obtained from all the 85 patients. 29 cases were given "excellent"; 48 were given "good". 6 patients experienced heavier low back pain, thus being classified as "fair". 2 cases with recurrence were given "poor".

Conclusions: PLF-PELD with specially designed instrument is a less invasive, effective and safe surgery for lumbar lateral recess stenosis with/without combined HDs.
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http://dx.doi.org/10.1016/j.clineuro.2016.02.008DOI Listing
April 2016

Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration.

Int J Biometeorol 2016 May 4;60(5):727-36. Epub 2015 Oct 4.

College of Agronomy, Heilongjiang Bayi Agricultural University, Daqing, 163319, China.

The global atmospheric CO(2) concentration is currently (2012) 393.1 μmol mol(-1), an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO(2) concentrations, an experiment was conducted using the Free Air CO(2) Enrichment (FACE )system. Two conventional japonica rice varieties (Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO(2) on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO(2) levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO(2) concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO(2) concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO(2) concentration increased enzyme activity expression and starch synthesis, affecting the final contents of starch and protein in the rice grains. Our results will provide a foundation for understanding the physiological mechanisms of rice yield under elevated atmospheric CO(2) concentrations.
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http://dx.doi.org/10.1007/s00484-015-1068-9DOI Listing
May 2016

The strategy and early clinical outcome of full-endoscopic L5/S1 discectomy through interlaminar approach.

Clin Neurol Neurosurg 2015 Jun 14;133:40-5. Epub 2015 Mar 14.

Department of Orthopaedics, The First Affiliated Hospital of General Hospital of Chinese People's Liberation Army, Beijing 100048, China.

Objective: To analyze the surgical strategy, safety and clinical outcome of full-endoscopic discectomy through interlaminar approach in the case of L5/S1 intervertebral disc excision.

Methods: From April 2011 to December 2011, 72 cases of intracanalicular non-contained disc herniations at L5/S1 level were treated with full-endoscopic discectomy through interlaminar approach. L5/S1 disc herniation was divided into three types according to position of herniated disc related to S1 nerve root: axilla type, ventral type and shoulder type. Axilla approach was selected for axilla type while shoulder approach was selected for ventral type and shoulder type. After operation, MRI was reexamined to evaluate the resection completeness of prolapsed disc material. Visual analog scales (VAS) of low back pain and sciatica, and Oswestry disability index (ODI) were recorded in certain preoperative and postoperative time points. MacNab scores were evaluated at the 12-month follow-up.

Results: All operations were completed without conversion to other surgical techniques. Average operation time was 45 min (20-80 min). Only one reoccurrence was revised with microendoscopic discectomy. No nerve injury and infection were complicated. Postoperative ODI and VAS of low back pain and sciatica were significantly decreased in each time point (P < 0.05). MacNab scores of 12-month follow-up include 44 excellent, 26 good, 1 fair and 1 poor.

Conclusion: With proper selection between axilla approach and shoulder approach according to the sites of prolapsed or sequestered disc materials, full-endoscopic L5/S1 discectomy through interlaminar approach is a safe, rational and effective minimally invasive spine surgery technique with excellent clinical short-term outcomes.
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http://dx.doi.org/10.1016/j.clineuro.2015.03.003DOI Listing
June 2015

Sustained and cancer cell targeted cytosolic delivery of Onconase results in potent antitumor effects.

J Control Release 2012 May;159(3):346-52

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.

The unfavorable pharmacokinetics and low tumor specificity hampered the potential clinical utility of Onconase, a promising modality in anticancer treatment with unique targets and novel mechanism of action. In this study, a modular and multi-stage drug delivery system (DDS) that can break down organ (renal accumulation), cellular (cancer cell specific uptake) and sub-cellular (endosomal escape) level barriers encountered by Onconase during its long journey from injection site to the cytoplasm of cancer cell was designed. Human serum albumin fusion extended the half-life of Onconase and significantly decreased its kidney accumulation. Epithelial cell adhesion molecular (EpCAM) specific antibody fragment appending enhanced binding and internalization of Onconase toward EpCAM positive cancer cell and increased its tumor accumulation and retention. Tethering Onconase to its carrier by cleavable disulfide linker prompted endosomal escape and restored its cytotoxicity. In vivo antitumor efficacy assay in human tumor xenograft model revealed that only when the entire organ, cellular and sub-cellular level barriers had been broken down, will Onconase turn into a potent antitumor agent.
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http://dx.doi.org/10.1016/j.jconrel.2012.02.019DOI Listing
May 2012

Balancing the pharmacokinetics and pharmacodynamics of interferon-α2b and human serum albumin fusion protein by proteolytic or reductive cleavage increases its in vivo therapeutic efficacy.

Mol Pharm 2012 Mar 1;9(3):664-70. Epub 2012 Feb 1.

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.

Human serum albumin (HSA) fusion (Albufusion) technology has evolved to be a general strategy to increase the in vivo half-lives of therapeutic proteins. However, because of the steric hindrance effect of HSA, conventional Albufusion technology improves the pharmacokinetics (PK) at the cost of pharmacodynamics (PD). To achieve balanced PK and PD of interferon-α2b (IFN-α2b) and HSA fusion protein, protease cleavage sites or disulfide linkage that enabled releasing of intact IFN-α2b with full activity was introduced between these two moieties. Nonreleasable and releasable fusion proteins showed similar cell surface receptor binding affinities; however, releasable fusion proteins exhibited release efficiency proportional increase of in vitro antiviral and antiproliferative activities. The release rate also had a profound impact on the in vivo pharmaceutical properties of fusion proteins. Releasable fusion proteins with intermediate release rate had the most balanced PK and PD, which translated into improved therapeutic efficacy in the HT29 human colon cancer xenograft model. Releasable Albufusion (rAlbufusion) allows tailored design of the PK/PD profile and greatly extends the utility of conventional Albufusion technology.
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http://dx.doi.org/10.1021/mp200347qDOI Listing
March 2012

Diagnosis of heart failure with preserved ejection fraction: which parameters and diagnostic strategies are more valuable?

Eur J Heart Fail 2011 Jul 20;13(7):737-45. Epub 2011 May 20.

Department of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.

Aims: There are no unified criteria for diagnosing heart failure with preserved ejection fraction (HFpEF). The aim of this study was to evaluate the present main diagnostic criteria and to discover which parameters and strategies are more valuable.

Methods And Results: Echocardiographic data and plasma N-terminal pro-brain natriuretic peptide  levels were assessed in a derivation cohort (n= 236) and a validation cohort (n= 98). Both cohorts included normal controls, patients with hypertensive heart disease without heart failure and patients with HFpEF. In the derivation cohort, the ratio of early mitral inflow velocity to tissue Doppler velocity at lateral mitral annulus (lateral E/e'≥12), left atrial volume index (LAVI≥34 mL/m(2)), and the difference between duration of reversed pulmonary vein atrial systole flow and duration of mitral A wave flow (Ard-Ad>30 ms) had the greatest diagnostic value among all the single parameters. A brief strategy that consisted of either: (i) lateral E/e'≥12; or (ii) 12>lateral E/e'≥8, with either LAVI≥34 mL/m(2) or Ard-Ad>30 ms, provided good diagnostic accuracy for identifying diastolic dysfunction in HFpEF, with a sensitivity of 77% and specificity of 81%. These observations were confirmed in the validation cohort.

Conclusion: Echocardiographic parameters including lateral E/e', LAVI, and Ard-Ad have the greatest value in diagnosing HFpEF. A brief strategy that included these three parameters had great diagnostic value and would be simple to use in clinic practice.
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http://dx.doi.org/10.1093/eurjhf/hfr053DOI Listing
July 2011

[Imagings of coronary total occlusion using dual-source computed tomography].

Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2010 Dec;32(6):677-82

Department of Radiology, Fourth Military Medical University, Xi'an 710032, China.

Objective: To analyze the imaging characteristics of coronary arteries with total occlusion (TO) lesions shown by dual-source computed tomography CT (DSCT) coronary angiography.

Method: The clinical data of patients who were diagnosed as coronary heart disease together with total occlusion lesions between March 2008 and March 2010 were retrospectively analyzed.

Results: In a cohort of 140 patients with 152 TO lesions, TO vessels in right coronary artery, left anterior descending artery, left circumflex coronary artery, or left main coronary artery were 68, 48, 20, and 1, respectively. Side branch TO were found in 15 vessels, in which 13 cases were found to be with TO in two coronary arteries. The length of TO was 5-58 mm, mean (16.8 ± 3.9) mm. TO lesions with a length ≥ 1 cm accounted for 91.4%. The appearance of stump in TO were abrupt occlusion (n=68) , blunt occlusion (n=64) , and mouse-tail occlusion (n=20) . Among all the TO lesions, 73% were soft plaque or mainly soft plaque together with minimal calcification. Mixed plaque and calcified plaque were less seen. Ten TO segments presented with dilated lumens were thrombogenesis. There were 25 TO segments angulated or tortuosity, which were most frequently shown in right coronary artery. DSCT only presented 3 TO with clear collateral vessels and no TO with bridge collateral vessels was shown.

Conclusions: DSCT can provide most necessary information of coronary TO lesions. Therefore, it can be used to guide surgeries on TO lesions and improve the success rates of surgeries.
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http://dx.doi.org/10.3881/j.issn.1000.503X.2010.06.018DOI Listing
December 2010

Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-alpha2b and HSA fusion protein increases its stability against mechanical and thermal stresses.

Eur J Pharm Biopharm 2009 Jun;72(2):405-11

Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing, People's Republic of China.

Interferon-alpha2b (IFN-alpha2b) and human serum albumin (HSA) fusion protein (IFN-alpha2b-HSA) is a promising long acting formulation of IFN-alpha2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-alpha2b-HSA was prone to disulfide-linked aggregation. The formation of aggregates was associated with an increase in immunogenicity in mice. The addition of non-ionic surfactant Tween 80 increased the stability of IFN-alpha2b-HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-alpha2b-HSA during quiescent storage. To increase the stability of IFN-alpha2b-HSA, the unpaired cysteine residue in this fusion protein was substituted with serine by site-directed mutagenesis. The resultant fusion protein was designated as IFN-alpha2b-HSA(C34S). IFN-alpha2b-HSA(C34S) had significant higher stability over IFN-alpha2b-HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 degrees C for 2 h, more than 90% of IFN-alpha2b-HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-alpha2b-HSA(C34S) increased less significantly after agitation. Pharmacokinetics studies in rats revealed that both fusion proteins had similar pharmacokinetic behavior, both with a half-life of about 50 h.
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http://dx.doi.org/10.1016/j.ejpb.2009.01.008DOI Listing
June 2009

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization.

FEMS Yeast Res 2009 Jun 16;9(4):591-9. Epub 2009 Mar 16.

Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing, China.

Onconase, an RNAse extracted from embryos of the Northern leopard frog (Rana pipiens), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an alpha-mating factor (alpha-MF)-prepro secretion signal, and an alpha-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L(-1) for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.
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http://dx.doi.org/10.1111/j.1567-1364.2009.00498.xDOI Listing
June 2009

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast.

J Biotechnol 2009 Jan 8;139(2):131-6. Epub 2008 Oct 8.

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.

Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43kDa and 66.2kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60h of induction during culture, it was found that the degradation product of around 66.2kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).
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http://dx.doi.org/10.1016/j.jbiotec.2008.09.006DOI Listing
January 2009

[Effects of 16-multi-detector row CT aortography with lower tube current].

Zhonghua Yi Xue Za Zhi 2008 Jun;88(21):1462-6

Department of Radiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.

Objective: To study the effects of 16-multi-detector row CT (MDCT) aortography with lower tube current in diagnosis of aortic diseases.

Methods: The study was conducted in 2 steps. In the first step, 58 patients with suspicious aortic disease or after operation on the aorta underwent 16-MDCT aortography for 70 times. Ten of them underwent scanning with conventional tube current of 175 mAs and the other 60 patients were divided into 3 groups according to their bogy weights: <65 kg group (n=20) receiving the lowest tube current of 25 mAs, 65-75 kg group (n=20) receiving the lowest tube current of 50 mAs, and >75 kg group (n=20) receiving the lowest tube current of 75 mAs. In the second step 60 patients with dissecting aneurysm, underwent 16-MDST aortography and were divided into 3 groups according to their body weight too: <65 kg group (n=20) receiving the lowest tube current of 50 mAs, 65-75 kg group (n=20) receiving the lowest tube current of 75 mAs, and >75 kg group (n=20) receiving the lowest tube current of 100 mAs, all 25 mAs more compared with the corresponding groups in the first step. The weighted CT dose index (CTDI), scanning length, and dose length produce (DLP) were recorded. The diagnostic accuracy rates of the images from the low dose groups were compared with those of the higher dose groups.

Results: The data of the first step showed that the CTDI values of the patients who received 25 mAs, 50 mAs, and 75 mAs tube current were 11.3%, 29.0%, and 42.7% that of the conventional tube current group (all P < 0.001) and the DLP values of the 3 low dose groups were also significantly lower than that of the conventional tube current group (all P < 0.001). The diagnostic accuracy rate of the 25 mAs, 50 mAs, and 75 mAs groups were 60% , 85%, and 85% that of the conventional tube current group. The results of the second step showed that the CTDI values of the 50 mAs, 75 mAs, and 100 mAs groups were 29.0%, 42.7%, and 57.3% that of the conventional tube current group respectively, and the DLP values of the 3 low dose groups were also significantly lower than that of the conventional group ( all P < 0. 001); and the 50 mAs, 75 mAs, and 100 mAs groups all showed good three-dimensional reconstruction imaging qualities, all with the diagnostic accuracy rate of 100%. The crossing section and three-dimensional images all showed excellent diagnostic image quality.

Conclusion: 16-MDCT aortography with the tube current at the doses 50 mAs to 100 mAs suffices to diagnose aortic diseases in patients with different body weights. Higher tube current should be used in dissecting aneurysm. The tube current at the dose of 100 mAs satisfies the imaging and diagnosing of all kinds of aortic diseases in the patients with any body weight.
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June 2008

Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization.

Appl Microbiol Biotechnol 2008 Nov 27;81(2):235-41. Epub 2008 Aug 27.

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai district, Beijing, 100071, People's Republic of China.

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha 2b fusion protein (HSA-IFN-alpha 2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha 2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha 2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha 2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20 degrees C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.
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http://dx.doi.org/10.1007/s00253-008-1666-0DOI Listing
November 2008

Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha2b fusion protein by linker engineering.

Protein Expr Purif 2008 Sep 16;61(1):73-7. Epub 2008 May 16.

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.

Previous studies in our laboratory have shown that when the N-terminus of interferon-alpha2b (IFN-alpha2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-alpha2b) was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-alpha2b was ascribed to the structural disturbance between HSA and IFN-alpha2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-alpha2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-alpha2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS-PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-alpha2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-alpha2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 degrees C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-alpha2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively.
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http://dx.doi.org/10.1016/j.pep.2008.04.013DOI Listing
September 2008

Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon-alpha2b fusion protein.

Yeast 2008 Apr;25(4):279-86

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.

In an attempt to increase the secretion capacity of Pichia pastoris (Pp), PpPMR1 gene was disrupted with GS115 as parent strain, and the resultant mutant was designated as Pppmr1. Pppmr1 displayed a Ca2+-dependent growth defect, which was consistent with the PMR1 mutation in other yeasts. HSA-L5-IFNalpha2b, a human serum albumin (HSA) and inferferon-alpha2b (IFNalpha2b) fusion protein with a flexible linker of 5 amino acid residues, was employed as a reporter to study the effects of PpPMR1 disruption on the secretion of heterologous protein. Because of its decreased viability after induction, Pppmr1 secreted more HSA-L5-IFNalpha2b only during the early phase (the first 15 hours) of induction. Although HSA-L5-IFNalpha2b secreted from GS115 and Pppmr1 had similar antiviral activity, the latter was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to aggregation at neutral to mild alkaline pH). Site-directed mutagenesis revealed that the heterogeneity of HSA-L5-IFNalpha2b secreted from Pppmr1 was originated from the incomplete disulphide bridge pairing between Cys1 and Cys98 of IFNalpha2b. To be secreted homogeneously from Pppmr1 and to be stable in aqueous solution, the linker of the fusion protein should be extended to 10 amino acid residues.
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http://dx.doi.org/10.1002/yea.1589DOI Listing
April 2008

Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation.

J Biotechnol 2007 Sep 29;131(3):245-52. Epub 2007 Apr 29.

Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.

Albuferon is a novel long-acting interferon resulted from the direct genetic fusion of human albumin and interferon-alpha2b (HSA-IFN-alpha2b). Albuferon, co-developed by Human Genome Sciences Inc. and Novartis, is currently in late stage development for the treatment of hepatitis C. It was unexpected that HSA-IFN-alpha2b secreted from Pichia pastoris migrated as doublets on non-reducing SDS-PAGE and was prone to form covalent aggregates in aqueous solution. The heterogeneity and instability of HSA-IFN-alpha2b lowered its recovery rate to about 10% and necessitated lyophilized formulation. Site-directed mutagenesis revealed that the heterogeneity and instability of HSA-IFN-alpha2b was caused by the incomplete disulfide bridge formation between Cys1 and Cys98 of IFN-alpha2b. To alleviate the structural perturbation of IFN-alpha2b by HSA, IFN-alpha2b-HSA fusion protein, in which IFN-alpha2b was located at the N-terminus, was created. IFN-alpha2b-HSA was shown to be homogeneous and stable at 37 degrees C for at least 10 days. The improved homogeneity and stability of IFN-alpha2b-HSA increased the recovery rate by 2.5-fold and made the development of stable solution formulation possible. In vitro antiviral assays showed that both fusion proteins retained the activity of IFN-alpha2b, and the EC(50) of HSA-IFN-alpha2b, and IFN-alpha2b-HSA was calculated to be 120+/-12.5, and 160+/-1 1.3ng/ml, respectively. The increased recovery rate and the possibility of solution formulation of IFN-alpha2b-HSA may compensate for its slightly decreased in vitro activity, and makes it to be a promising therapeutic agent that deserves further evaluation.
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http://dx.doi.org/10.1016/j.jbiotec.2007.04.016DOI Listing
September 2007

Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response.

Vaccine 2007 May 28;25(22):4478-86. Epub 2007 Mar 28.

Department of Microbiological Engineering, Beijing Institute of Biotechnology, 20 Dongdaije Street, Fengtai District, Beijing 100071, China.

Many studies have provided evidence that heat shock protein 65 (Hsp65) can elicit potent specific cellular adaptive immune responses (e.g. CD8(+) cytotoxic T-cell effectors or classic CTLs) based on their ability to chaperone antigenic peptides. Hsp65 is thus an effective carrier for heterologous peptide epitopes for therapeutic vaccines against cancer or chronic infectious diseases. The core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, and functions as both a T-cell-dependent and a T-cell-independent antigen. Therefore, HBcAg may be a promising candidate target for therapeutic vaccine control of chronic HBV infection. Here, a chimeric protein, Hsp65Bc, was created by fusing the HBcAg sequence to the carboxyl terminus of the Hsp65 sequence in E. coli. Analysis of its antigenicity and immunogenicity revealed that HBc epitopes are surface accessible. Hsp65Bc induced moderate anti-HBc immune responses as well as a strong specific T-cell response in BALB/c mice. These results indicate that Hsp65Bc may have potential as a vaccine for treatment of HBV chronic infection.
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http://dx.doi.org/10.1016/j.vaccine.2007.03.020DOI Listing
May 2007

Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins.

Biotechnol Prog 2006 Jul-Aug;22(4):1090-5

Beijing Institute of Biotechnology, China.

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.
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http://dx.doi.org/10.1021/bp060019rDOI Listing
October 2007

Construction and evaluation of anti-gastrin immunogen based on P64K protein.

World J Gastroenterol 2006 Apr;12(13):2040-6

Department of Microbiologic Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.

Aim: To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect.

Methods: G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed.

Results: G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84(th) day after the first immunization, the titer of antibody against cross-linked protein reached 51,200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480.

Conclusion: The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4087682PMC
http://dx.doi.org/10.3748/wjg.v12.i13.2040DOI Listing
April 2006