Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

Production and characterization of monoclonal antibodies to a grouper iridovirus

A panel of six monoclonal antibodies (mAbs) against a grouper iridovirus (SGIV) were produced by immunization of Balb/c mice with purified virus preparations. Isotyping test revealed all the mAbs were IgG1. None of the mAbs possessed the ability to neutralize SGIV in cell cultures. Western blot showed that 4 mAbs reacted with 2 SGIV proteins at molecular mass of approximately 100 and 117 kDa in gradient-purified virus. Immunofluorescent studies showed that the two specific viral proteins VP100 and VP117 were localized within virus assembly sites in the cytoplasm of SGIV-infected grouper cells (GP). Fractionations of the iridovirus in a 20-60% sucrose gradient were detected successfully by all the six mAbs using immunodot blot. An antigen-capture enzyme-linked immunosorbent assay (ELISA) system, based on the use of mAb 7E11 for capture and a rabbit polyclonal antibody to SGIV for detection was developed. SGIV antigen was detected in gradient-purified virus and virus-infected grouper blood. These novel mAbs will facilitate the development of more specific and standardized diagnostic techniques for marine fish iridovirus.     

Thermal strain and fluid balance during a 72-km military route march in a field setting

Introduction:A physiological profiling study was conducted to evaluate thermal strain as well as fluid and electrolyte balances on heat-acclimatised men performing a 72-km route march in a field setting.Methods:38 male soldiers (age range 18–23 years) participated in the study, as part of a cohort that marched for 72 km, with loads for about 26 hours. Core temperature and heart rate sensors were used for the duration of the march. Fluid and food intake and output were monitored for the duration of the march. Blood samples were taken one day before the march (pre-march), immediately after the march (Post 1) and on the 15^th day after the march to ascertain recovery (Post 2) to assess fluid and electrolyte profiles.Results:Mean core temperature was within safe limits, ranging from 37.1 to 38.1°C throughout the march. There was an overall decrease in serum sodium levels, a decline in serum sodium concentration in 28 participants and three instances of hyponatraemia (serum sodium concentration <135 mmol/L).Conclusion:Our study found low thermal strain heat-acclimatised individuals during a 72-km route march. However, there was an overall decrease in serum sodium levels, even when the participants were allowed to drink ad libitum. Challenges of exercise-associated hyponatraemia during prolonged activities remain to be addressed.     

Polypyrrole/tannin biobased nanocomposite with enhanced electrochemical and physical properties

In this research, tannin (TA) extracted from Acacia mangium and a cationic surfactant, cetyltrimethylammonium bromide (CTAB), were used to modify and enhance the physical and electrochemical properties of a polypyrrole (PPy) composite. Brunauer–Emmett–Teller (BET) analysis presented a higher degree of surface area and porosity for the PPy/TA/CTAB nanocomposite. A highly porous and rod like structure with a lumpy surface was observed for PPy/TA prepared in the presence of CTAB by Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). Cyclic voltammograms of the modified SPE electrode using PPy/TA/CTAB displayed an enhanced current response compared to the electrode modified with only PPy or PPy/TA. Electrochemical Impedance Spectroscopy (EIS) exhibited a lower value of charge transfer resistance (R_ct) and higher electron transfer for the modified electrode, making the nanocomposite a promising candidate for biosensor application.

Tannin (TA) extracted from Acacia mangium and a cationic surfactant, cetyltrimethylammonium bromide (CTAB), were used to modify and enhance the physical and electrochemical properties of polypyrrole (PPy) composite.     

Emergency Presenting Colon Cancer Is an Independent Predictor of Adverse Disease-Free Survival

Twenty percent of colon cancers present as an emergency. However, the association between emergency presentation and disease-free survival (DFS) remains uncertain. Consecutive patients who underwent elective (CC) and emergent (eCC) resection for colon cancer were included in the analysis. Survival outcomes were compared between the 2 groups in univariate/multivariate analyses. A total of 439 patients underwent colonic resection for colon cancer during the interval 2000−2010; 97 (22.1%) presented as an emergency. eCC tumors were more often located at the splenic flexure (P = 0.017) and descending colon (P = 0.004). The eCC group displayed features of more advanced disease with a higher proportion of T4 (P = 0.009), N2 tumors (P < 0.01) and lymphovascular invasion (P< 0.01). eCC was associated with adverse locoregional recurrence (P = 0.02) and adverse DFS (P < 0.01 ) on univariate analysis. eCC remained an independent predictor of adverse locoregional recurrence (HR 1.86, 95% CI 1.50–3.30, P = 0.03) and DFS (HR 1.30, 95% CI 0.88–1.92, P = 0.05) on multivariate analysis. eCC was not associated with adverse overall survival and systemic recurrence. eCC is an independent predictor of adverse locoregional recurrence and DFS.Key words: Emergency presentation, Colon cancer, Disease free survival, Locoregional recurrenceColorectal cancer (CRC) is a significant cause of mortality, with over 40,000 new cases diagnosed annually in the UK contributing to over 16,000 deaths (Bowel Cancer UK).^1,2 Up to 20% of colon cancers (CC) present as an emergency (eCC) necessitating emergent surgery.^3,4 Although eCC has been shown to be associated with poorer overall survival (OS), much discrepancy exists in the literature regarding its association with disease-free survival (DFS).⁵⁻⁷Studies reporting the oncologic outcomes of CRC presenting as an emergency consist of heterogeneous populations of patients with colon and rectal cancers.^3,5 Colon and rectal cancers are 2 distinct entities with different molecular, clinical, pathologic, and biologic characteristics and treatment modalities.^8,4,9−11 Since the incorporation of combined multimodal treatment and total mesorectal excision the disparity in OS and DFS between colon and rectal cancer has increased.¹²⁻¹⁷ Rectal cancer patients may alter the impression of outcomes in emergency presenting colon cancer. Consequently, previous studies assessing outcomes in eCC may be flawed. Furthermore, the negative impact of eCC has previously been attributed to immediate postoperative complications with an inpatient hospital mortality of approximately 15%. Inclusion of such cases in studies assessing long-term outcomes may have overestimated the negative impact of eCC.¹⁸The aim of the current study was to determine the association between eCC and disease-free/overall survival.     

Increased use of multidisciplinary treatment modalities adds little to the outcome of rectal cancer treated by optimal total mesorectal excision

Purpose

Total mesorectal excision (TME) is the standard surgical treatment for rectal cancer. The roles of chemotherapy and radiotherapy have become more defined, accompanied by improvements in preoperative staging and histopathological assessment. We analyse our ongoing results in the light of changing patterns of treatment over consecutive time periods.

Methods

In total, 151 consecutive patients underwent potentially curative rectal excision for cancer in a single institution. Management and outcomes were compared between 1993–1999 and 2000–2007 which corresponded with the restructuring of the regional oncological services.

Results

We found an increase in patients treated with neoadjuvant chemoradiotherapy after 1999 (20/89 vs 1/62, p?p?=?0.037). The locoregional recurrence rate was 5.3%. The rates were not significantly different between the two study periods [4/89 (4.5%) 1999–2007 vs 4/62 (6.5%) 1993–1999, p?=?0.597]. There was no statistical difference in overall or disease-free survival in the time periods examined.

Conclusions

Increasing use of neoadjuvant therapy and concomitant improvement in lymph node assessment did not translate into a concurrent reduction in the local recurrence, disease-free and overall survival rates. Our results demonstrate the enduring benefit of specialist training in TME in the outcome of rectal cancer surgery. This observational study suggests that low local recurrence rates are surrogate markers for improved overall and disease-free survival. Multidisciplinary team practice should be examined and made cost effective according to the individual unit’s local recurrence rate in the light of this and other reports.