Direct Recovery of Recombinant Hepatitis B Core Antigen from Unclarified Escherichia Coli Feedstock Using Expanded Bed Adsorption Chromatography

The capsid of hepatitis B virus (HBV), which consists of hepatitis B core antigen (HBcAg) has become one of the most frequently studied viral-like-particle (VLP) for the display of foreign epitopes. Many studies have been carried out to purify this capsid. However, the conventional method of purification requires multiple steps of operation, which could lead to excessive product loss and high production costs. Therefore, it is of importance to develop a fast and cost effective protein recovery method such as expanded bed adsorption chromatography (EBAC) to ensure a better and efficient recovery of protein, especially in large scale downstream process. In this study, thermal treatment of the Escherichia coli cell feedstock at 60oC for 30 min prior to solid removal in the conventional method has resulted in 1.4 times and 18% higher in purity and recovery yield respectively compared to non-heat-treated feedstock. In direct capture of HBcAg from unclarified feedstock using the STREAMLINE DEAE (weak anion-exchangers) in batch adsorption, heat treatment at 60oC for 45 min has increased the recovery yield and purity by 2.3 and 3.8 times respectively compared with non-heat-treated feedstock. When these conditions were applied in large scale purification of HBcAg via EBAC, the yield and purity have increased by 1.2 and 1.8 folds respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and anion-exchanger. The present study has also demonstrated that purification of HBcAg from heat-treated unclarified feedstock was most efficient when EBAC operation using FastlineTM 20 contactor was operated at constant velocity (127.9 cm/h) in feedstock containing 5% of biomass. Although the current study showed that heat-treatment of unclarified feedstock could increase the purity of HBcAg and reduced non-specific binding of contaminant onto Streamline DEAE, the purity obtained was lower compared with that purified using conventional methods. Therefore, development of an affinity adsorbent using M13 phage bearing a disulfide constrained heptapeptide at the gpIII protein coat with the sequence, C-WSFFSNI-C as the ligand has been carried out in this study. M13 phage immobilised onto Streamline Base Matrix via epoxy activation was used in direct capture of HBcAg from unclarified feedstock via two different modes of EBAC operations; typical single pass operation and modified EBAC operation with recirculation of feedstock. Higher yield of HBcAg was obtained using modified EBAC operation due to increase in protein residence time in the column, however, the purity was reduced by 15% compared with typical EBAC operation, which could be due to diffusion of contaminants into the internal volume of the macroporous adsorbents. Although the purity of HBcAg recovered using M13 phage ligand adsorbents was higher (70-80%) but the yield was lower compared with that purified using anion-exchanger. Therefore, this study showed that peptide displayed on M13 phage can be employed as an affinity ligand in direct capture of HBcAg from unclarified feedstock using EBAC. When analysed with ELISA, the antigenicity of HBcAg purified using both adsorbents in EBAC was still preserved

Effect of Channa Striatus and Curcuma Longa in Experimentally-Induced Osteoarthritis in Rabbits

Channa striatus and Curcuma longa are two well known natural products that have long been used in treating various kinds of ailments. Channa striatus is high in essential amino acids and fatty acids that played an important role in wound healing as well as in anti-nociceptive activities. Curcuma longa on the other hand, contains an active compound called curcuminoids that are responsible for its anti-inflammatory, anti-oxidant and anti-cancer properties. Therefore, in this study, Channa striatus and Curcuma longa extracts were used in the treatment of experimentally induced Osteoarthritis (OA) in rabbits. OA was induced on the right stifle joint of the rabbits in the treatment and the negative control groups by transecting the anterior cruciate ligaments. These animals were left for 8 weeks to develop OA. Radiography and ultrasonography were performed on the induced joints to determine the development of OA prior to Channa striatus and Curcuma Jonga treatments. During the progression of OA, the induced joints began to show sign of OA development as early as the 2nd week after induction of OA as observed in ultrasonograph. Slight joint space narrowing, which reflect the deteriorating articular cartilage was detected by the ultrasonography as early as the 2nd week post induction. On the 3rd week after the induction of OA, ultrasonography was able to detect significant joint space narrowing and total diminution of joint space on the 4th week post induction. In addition to that, irregular joint surface has developed in the induced joints as seen on the ultrasonograph taken on the 5th and the 6th week post induction. Apart from these, other structures such as the infra patellar fat, the patellar ligament and the synovial membrane in the induced joints also underwent osteoarthritic changes as seen in ultrasonographs. On the 8th week post induction there was a significant periarticular soft tissue swelling detected by radiography and ultrasonography. Soft tissue swelling detected on the radiographs was seen as an increased radiopacity area around the joint. In ultrasonographs, the swelling of the joint could be observed as an increased distance between the surface of joint and the skin compared to the normal uninduced joints. On the 9th week of treatment, a significant reduction of soft tissue swelling was observed on Channa striatusand Curcuma longa- treated joints compared to the untreated joints. Although the treatments were effective in reducing inflammations and swelling, these extracts d id not exhibit any improvement on other structures of the joints. Extra bone formation and diminution of the joint space were observed on both radiographs and ultrasonographs on the 9th week of treatment. These similar changes were further confirmed with the gross findings on the opened joints upon euthanasia. In the immunohistochemistry study, synovial membrane biopsies from the normal, treated and negative control joints were obtained to study the general innervation of the synovial membrane. The immunoreactive fibres stained against PGP 9.5, CGRP and NPY antisera were not detected in the control joints compared to the normal synovial membrane. The synovial membrane from the untreated joints was heavily infiltrated with inflammatory cells, which may be account for the diminished immunoreactive nerve fibres from the synovial membrane. However, the number of immunoreactive nerve fibres detected in the synovial membranes from Channa striatus and Curcuma longa treated joints was higher than in the synovial membrane from the control untreated joint. They exhibited a similar distribution to the nerve fib res found in normal synovial membrane but less numerous. Therefore, the present study showed that both Channa striatus and Curcuma tonga extracts showed good signs of healing in OA and these extracts can be used as a good alternative treatment in OA

Effect of different operating modes and biomass concentrations on the recovery of recombinant hepatitis B core antigen from thermal-treated unclarified Escherichia coli feedstock

Expanded bed adsorption chromatography (EBAC) is a single pass operation that has been used as primary capture step in various protein purifications. The most common problem in EBAC is often associated with successful formation of a stable fluidized bed during the absorption stage, which is critically dependent on parameters such as liquid velocity, bed height, particle (adsorbent) size and density as well as design of column and type of flow distributor. In this study, residence time distribution (RTD) test using acetone as non-binding tracer acetone was performed to evaluate liquid dispersion characteristics of the EBAC system. A high B(o) number was obtained indicating the liquid dispersion in the system employed is very minimal and the liquid flow within the bed was close to plug flow, which mimics a packed bed chromatography system. Evaluation on the effect of flow velocities and bed height on the performance of Streamline DEAE using feedstock containing heat-treated crude Escherichia coli homogenate of different biomass concentrations was carried out in this study. The advantages and disadvantages as well as the problems encountered during recovery of HBcAg with aforementioned parameters are also discussed in this paper

N-terminally His-tagged hepatitis B core antigens: construction, expression, purification and antigenicity

The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg

Production of fusion m13 phage bearing the di-sulphide constrained peptide sequence (C-WSFFSNI-C) that interacts with hepatitis B core antigen

Effects of pH, temperature, and level of mixing on the production of fusion M13 phage bearing the peptide sequence (C-WSFFSNI-C) that interacts with HBcAg were investigated in this study. The optimum pH for the phage production was achieved at pH 7, followed by pH 6 and 8. The highest fusion phage titre was obtained at growth temperature of 37°C, followed by 27 and 42°C. The rotational speed at 250 rpm was the optimal mixing level for the phage production. Further increase of rotational speed to 300 rpm has reduced the phage production to a level lower than that obtained at 200 rpm. The results also showed that the propagation of fusion M13 phage has greatly affected the growth of Escherichia coli ER 2738. The viability of the phage produced with the current method was then determined using phage titre and dot-blot assays

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research