Development of a Novel Antibacterial Peptide, PAM-5, via Combination of Phage Display Selection and Computer-Assisted Modification.

Antibacterial peptides (ABPs) have been proposed as potential candidates for alternative antibacterial agents due to the extensive dissemination of antibiotic resistance. However, ABP isolation from natural resources can be tedious without consistent yield. Moreover, many natural ABPs are not developed for clinical application due to potential toxicity to mammalian cells. Therefore, the objective of this study was to develop a potent ABP with minimal toxicity via phage display selection followed by computer-assisted modification. Briefly, a 12-mer phage-displayed peptide library was used to isolate peptides that bound to the cell surface of Pseudomonas aeruginosa with high affinity. The affinity-selected peptide with the highest selection frequency was modified to PAM-5 (KWKWRPLKRKLVLRM) with enhanced antibacterial features by using an online peptide database. Using in vitro microbroth dilution assay, PAM-5 was shown to be active against a panel of Gram-negative bacteria and selected Gram-positive bacteria. Interestingly, the peptide was stable in human plasma by exhibiting a similar bactericidal effect via ex vivo assay. Scanning electron microscopy and SYTOX Green uptake assay revealed that PAM-5 was able to cause membrane disruption and permeabilization of the bacteria. Additionally, the peptide was also able to bind to bacterial DNA as demonstrated by gel retardation assay. In the time-kill assay, PAM-5 was shown to kill the bacteria rapidly in 10 min. More importantly, PAM-5 was non-cytotoxic to Vero cells and non-haemolytic to human erythrocytes at all concentrations tested for the antibacterial assays. Thus, this study showed that the combination of phage display screening and computer-assisted modification could be used to develop potent novel ABPs, and PAM-5 derived from these approaches is worth to be further elucidated for its potential clinical use.

Effect of cell microenvironment on the drug sensitivity of hepatocellular cancer cells.

The native hepatocellular cancer (HCC) microenvironment is characterized by more hypoxic, hypoglycemic, and acidic conditions than those used in standard cell culture. This study aimed to investigate whether HCC cells cultured in more native conditions have an altered phenotype and drug sensitivity compared to those cultured in standard conditions. Six HCC cell lines were cultured in "standard" (21% O2, 25 mM glucose) or more "native" (1% O2, 5 mM glucose, 10 mM lactate) conditions. Cells were assessed for growth rates, cell cycle distribution, relevant metabolite and protein levels, genome-wide gene expression, mitochondrial DNA sequence and sensitivity to relevant drugs. Many differences in cellular and molecular phenotypes and drug sensitivity were observed between the cells. HCC cells cultured in native conditions had slower doubling times, increased HK2 and GLUT, lower PHDA and ATP levels, and mutations in mitochondrial DNA. Thirty-one genes, including the hypoxia-associated NDRG1, were differentially expressed between the cells. HCC patients in The Cancer Genome Atlas (TCGA) with tumors with a high score based on these 31 genes had a poorer prognosis than those with a low score (p = 0.002). From 90 comparisons of drug sensitivity, increased resistance and sensitivity for cells cultured in native conditions was observed in 14 (16%) and 8 (9%) comparisons respectively. In conclusion, cells cultured in more native conditions can have a more glycolytic and aggressive phenotype and varied drug sensitivity to those cultured in standard conditions, and may provide new insights to understanding tumor biology and drug development.

Emergency department resuscitative thoracotomy at an adult major trauma centre: Outcomes following a training programme with standardised indications.

OBJECTIVE: The objective of this study was to report the procedural incidence and patient outcomes after the 2009 introduction of an institutional resuscitative thoracotomy (RT) programme. Emergency physicians, general surgeons and emergency nursing trauma team members were trained to perform RT on thoracic trauma patients with an unresponsive systolic blood pressure (SBP) <70 mmHg within 30 min of arrival, prior to cardiothoracic team back-up. METHODS: A retrospective cohort study was conducted on patients who underwent RT from 2009 to 2017. The primary outcome measures were the incidence of the procedure and patients' survival to hospital discharge. Variables associated with survival were assessed using univariable logistic regression analyses. RESULTS: There were 12 399 major trauma patients, including 7657 with major thoracic trauma and 315 presenting with SBP <70 mmHg. There were 32 RTs performed (incidence of 0.4%; 95% confidence interval [CI] 0.3-0.6) among patients with major thoracic trauma and 10.2% (99% CI 7.3-13.4) among patients with major thoracic trauma and SBP <70 mmHg. There were eight (25%; 95% CI 13.2-42.1) survivors to hospital discharge and no late mortality (mean follow-up 2.8 years). Survival was significantly associated with the procedure performed within 30 min of arrival (odds ratio 0.09; 95% CI 0.01-0.67) while mortality was associated with the procedure being performed in the setting of traumatic cardiac arrest (odds ratio 18.3; 95% CI 2.4-140.4). CONCLUSIONS: A formal training and credentialing programme was associated with a low incidence of the procedure, yet achieved a survival rate of 25%, which is comparable to other reported literature.

Acquired resistance to PI3K/mTOR inhibition is associated with mitochondrial DNA mutation and glycolysis.

Acquired resistance (AQR) to drug treatment occurs frequently in cancer patients and remains an impediment to successful therapy. The aim of this study was to gain insight into how AQR arises following the application of PI3K/mTOR inhibitors. H1975 lung cancer cells with EGFR T790M mutations that confer resistance to EGFR inhibitors underwent prolonged treatment with the PI3K/mTOR inhibitor, BEZ235. Monoclonal cells with stable and increased resistance to BEZ235 were obtained after 8 months treatment. These AQR clones showed class-specific resistance to PI3K/mTOR inhibitors, reduced G1 cell cycle arrest and impedance of migration following PI3K/mTOR inhibition, reduced PTEN expression and increased Akt and S6RP phosphorylation. Transcriptome analysis revealed the AQR clones had increased expression of the metabolite transporters SLC16A9 and SLC16A7, suggestive of altered cell metabolism. Subsequent experiments revealed that AQR clones possess features consistent with elevated glycolysis, including increased levels of glucose, lactate, glutamine, glucose dependence, GLUT1 expression, and rates of post-glucose extracellular acidification, and decreased levels of reactive oxygen species and rates of oxygen consumption. Combination treatment of BEZ235 with the glycolysis inhibitor 3-bromopyruvate was synergistic in AQR clones, but only additive in parental cells. DNA sequencing revealed the presence of a mitochondrial DNA (mtDNA) MT-C01 variant in AQR but not parental cells. Depletion of mitochondrial DNA in parental cells induced resistance to BEZ235 and other PI3K/mTOR inhibitors, and was accompanied by increased glycolysis. The results of this study provide the first evidence that a metabolic switch associated with mtDNA mutation can be an underlying mechanism for AQR.

Cryoprecipitate administration after trauma.

OBJECTIVES: Haemorrhage remains among the most preventable causes of trauma death. Massive transfusion protocols, as part of 'haemostatic resuscitation', have been implemented in most trauma centres. Relative to the attention to the ideal ratio of red blood cells to fresh frozen plasma and platelets, cryoprecipitate treatment has been infrequently discussed. We aimed to outline the use of cryoprecipitate during trauma resuscitation and analyse outcomes in patients who received cryoprecipitate after hypofibrinogenaemia detection. METHODS: A retrospective review of registry data on all major trauma patients (Injury Severity Score>15) presenting to a level I trauma centre over a 4-year period (2008-2011) was conducted. We selected all patients who had received cryoprecipitate and then analysed patients who had received cryoprecipitate following the detection of hypofibrinogenaemia (<1.0 g/l). Mortality at hospital discharge among hypofibrinogenaemic patients who had received cryoprecipitate was compared with that among patients who had not received cryoprecipitate. RESULTS: Of 3996 trauma patients, 3571 had fibrinogen levels recorded. Most patients (n=3517, 98.5%) had initial fibrinogen counts of 1.0 g/l or higher, and cryoprecipitate was administered to a small proportion of these patients (n=126, 3.6%). Of the 54 patients with hypofibrinogenaemia on arrival, one patient died immediately and was excluded from further analysis. Of the 53 patients, 30 received cryoprecipitate and 28/53 died (53%). There was no difference in mortality between those who had received and those who had not received cryoprecipitate (14/30 vs. 14/23, P=0.31). CONCLUSION: Administration of cryoprecipitate was uncommon during trauma resuscitation, even among patients with hypofibrinogenaemia on presentation. This study provides no evidence towards improved outcomes from administration of cryoprecipitate.

Ultra-low-voltage CMOS-based current bleeding mixer with high LO-RF isolation.

This journal presents an ultra-low-voltage current bleeding mixer with high LO-RF port-to-port isolation, implemented on 0.13 µm standard CMOS technology for ZigBee application. The architecture compliments a modified current bleeding topology, consisting of NMOS-based current bleeding transistor, PMOS-based switching stage, and integrated inductors achieving low-voltage operation and high LO-RF isolation. The mixer exhibits a conversion gain of 7.5 dB at the radio frequency (RF) of 2.4 GHz, an input third-order intercept point (IIP3) of 1 dBm, and a LO-RF isolation measured to 60 dB. The DC power consumption is 572 µW at supply voltage of 0.45 V, while consuming a chip area of 0.97 × 0.88 mm(2).

50 MHz-10 GHz low-power resistive feedback current-reuse mixer with inductive peaking for cognitive radio receiver.

A low-power wideband mixer is designed and implemented in 0.13 µm standard CMOS technology based on resistive feedback current-reuse (RFCR) configuration for the application of cognitive radio receiver. The proposed RFCR architecture incorporates an inductive peaking technique to compensate for gain roll-off at high frequency while enhancing the bandwidth. A complementary current-reuse technique is used between transconductance and IF stages to boost the conversion gain without additional power consumption by reusing the DC bias current of the LO stage. This downconversion double-balanced mixer exhibits a high and flat conversion gain (CG) of 14.9 ± 1.4 dB and a noise figure (NF) better than 12.8 dB. The maximum input 1-dB compression point (P1dB) and maximum input third-order intercept point (IIP3) are -13.6 dBm and -4.5 dBm, respectively, over the desired frequency ranging from 50 MHz to 10 GHz. The proposed circuit operates down to a supply headroom of 1 V with a low-power consumption of 3.5 mW.

Low power upconversion mixer for medical remote sensing.

This work presents the design of a low power upconversion mixer adapted in medical remote sensing such as wireless endoscopy application. The proposed upconversion mixer operates in ISM band of 433 MHz. With the carrier power of -5 dBm, the proposed mixer has an output inferred 1 dB compression point of -0.5 dBm with a corresponding output third-order intercept point (OIP3) of 7.1 dBm. The design of the upconversion mixer is realized on CMOS 0.13 µm platform, with a current consumption of 594 µA at supply voltage headroom of 1.2 V.

Detection of Entamoeba histolytica in experimentally induced amoebic liver abscess: comparison of three staining methods.

OBJECTIVE: To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster. METHODS: Amoebic liver abscess was experimentally induced in a hamster by injecting 1 × 10(6) of axenically cultured virulent E. histolytica trophozoites (HM1-IMSS strain) into the portal vein. After a week post-inoculation, the hamster was sacrificed and the liver tissue sections were stained with H&E, PAS and IHC stains to detect the amoebic trophozoite. RESULTS: The three stains revealed tissue necrosis and amoebic trophozoites, but with varying clarity. H&E and PAS stained the trophozoites pink and magenta, respectively, however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology. On the other hand, IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues. CONCLUSIONS: It can be concluded that out of the three stains, IHC is the best for identification of E. histolytica trophozoites in tissue sections.

Detection of Entamoeba histolytica in experimentally induced amoebic liver abscess: comparison of three staining methods

<p><b>OBJECTIVE</b>To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster.</p><p><b>METHODS</b>Amoebic liver abscess was experimentally induced in a hamster by injecting 1 × 10(6) of axenically cultured virulent E. histolytica trophozoites (HM1-IMSS strain) into the portal vein. After a week post-inoculation, the hamster was sacrificed and the liver tissue sections were stained with H&E, PAS and IHC stains to detect the amoebic trophozoite.</p><p><b>RESULTS</b>The three stains revealed tissue necrosis and amoebic trophozoites, but with varying clarity. H&E and PAS stained the trophozoites pink and magenta, respectively, however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology. On the other hand, IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues.</p><p><b>CONCLUSIONS</b>It can be concluded that out of the three stains, IHC is the best for identification of E. histolytica trophozoites in tissue sections.</p>

The dawn of emergency medicine in Vietnam.

In 2009 emergency medicine had not been officially established as a specialty in Vietnam. As a result of a non-government organization identifying the need to improve the delivery of emergency care, the Vietnam2010 Symposium in Emergency Medicine was held in Hue in March 2010. This involved 1 week of activity including: an Emergency Medicine Conference, providing lectures and practical workshops in topics of emergency medicine; a Deans' Conference, dedicated to the development of emergency medicine as a specialty; a Disaster and EMS Conference; and an Emergency Nursing Conference. Vietnam2010 was a high impact event and was successful in raising the profile of emergency medicine. It formalized key international linkages, showcased the role of the knowledge and skills relevant to emergency care and provided the impetus for emergency medicine specialization in Vietnam. A consensus document committing to the development of emergency medicine as a specialty in Vietnam was signed by multiple national and international governmental, university and emergency medicine representatives. Challenges included a tendency for international flagbearers from mature systems to promote the specialty according to local expectations, with a consequent emphasis on vertical specialty topics and on technology, and the running of medical and nursing conferences separately. Vietnam now needs a medium-term plan to develop the specialty to ensure these initial steps are translated into a sustainable capacity to provide emergency care nationally.

Emergency physician credentialing for resuscitative thoracotomy for trauma.

OBJECTIVE: A low case incidence and variable skill level prompted the development of a credentialing programme and specific surgical training in resuscitative thoracotomy for emergency physicians at The Alfred, a Level 1 Adult Victorian Major Trauma Service. METHODS: A review of the incidence of traumatic pericardial tamponade and the objectives of resuscitative thoracotomy were undertaken. RESULTS: A training programme involving pre-reading of a 17 page teaching manual, a 40 min didactic lecture and a 2 h surgical skills station using anaesthetized pigs were developed. The specific indication for resuscitative thoracotomy for this programme is ultrasound demonstrated cardiac tamponade secondary to blunt or penetrating truncal trauma in a haemodynamically unstable patient with a systolic blood pressure of less than 70 mmHg despite pleural decompression and intravenous volume replacement. Cardiac electrical activity must be present. The primary aims of resuscitative thoracotomy taught are release of cardiac tamponade, control of haemorrhage and access for internal cardiac massage. CONCLUSION: Emergency physicians working in high-volume Trauma Centres are expected to diagnose cardiac tamponade and on occasion decompress the pericardium. Specific training in the procedure should be undertaken.

Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae.

Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.

Comparison of histopathological features of Vibrio cholerae O1 El Tor and O139 Bengal infections in rabbit intestinal mucosa.

Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.

Emergency medicine in Sri Lanka: the inevitable evolution to a new specialty.

Sri Lanka is a low-income country with a relatively advanced, equitable and accessible health-care system offered to its 20 million populations free of charge through a national pro-poor health policy. Its weaknesses in emergency services, however, surfaced in 2004 when it faced the Tsunami, the worst natural disaster of the world of the 21st century. Since then, the local health community with the assistance of the government and foreign aid agencies have embarked on a path to establish emergency services, improve its preparedness for disaster management and establishment of emergency medicine training. The present article traces this path and how it is evolving in the country.

Silica coating of nanoparticles by the sonogel process.

A modified aqueous sol-gel route was developed using ultrasonic power for the silica coating of indium tin oxide (ITO) nanoparticles. In this approach, organosilane with an amino functional group was first used to cover the surface of as-received nanoparticles. Subsequent silica coating was initiated and sustained under power ultrasound irradiation in an aqueous mixture of surface-treated particles and epoxy silane. This process resulted in a thin but homogeneous coverage of silica on the particle surface. Particles coated with a layer of silica show better dispersability in aqueous and organic media compared with the untreated powder. Samples were characterized by high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), and the zeta potential.

End-of-life decisions and palliative care in a children's hospital.

OBJECTIVE: To investigate clinical and demographic factors affecting the nature of end-of-life decisions and pediatric palliative care. DESIGN: Charts of 236 expired children were retrospectively reviewed for presence of endof- life care (EOLC) discussions and spiritual support, the nature of EOLC decisions, and the degree of opioid analgesics (OA) and sedatives (SDT) administration. RESULTS: Approximately 60% of patients had EOLC discussion, of whom 87.4% obtained an EOLC decision, mostly opting for withholding therapy (68.8%). Presence of EOLC discussion was associated with a longer hospital stay (univariate analyses: odds ratio [OR] = 1.9; p < 0.029), higher number of failed organs (OR = 2.5; p < 0.003), chronic illnesses (OR = 2.4; p < 0.002), spiritual support (OR = 1.8; p < 0.028) and respiratory diseases (OR = 3.1; p < 0.0006). Younger patients and those with higher number of failed organs were more likely to have withdrawal of therapy (OR = 10.9 and 6.0; p < 0.0001 and <0.002, respectively), whereas patients with chronic illness opted for withholding of therapy (OR = 3.1; p < 0.006). Spiritual support was associated with higher use of both OA and SDT (OR = 1.9 and 2.3; p < 0.014 and p < 0.005, respectively). Younger patients received less OA and SDT (OR = 0.2 and 0.4, respectively; p < 0.0001). Multivariate analyses showed that EOLC discussion is associated with higher use of OA and SDT (OR = 4.4 and 4.2; p < 0.00001 and p < 0.0001, respectively), whereas younger age is associated with withdrawal of therapy (OR = 8.3; p < 0.0005) and lower use of SDT (OR = 0.23; p < 0.0001). CONCLUSIONS: Patterns of care at the end of life vary in children with differing clinical and demographic characteristics. Because EOLC discussions are associated with greater focus on palliative care, strategies to enhance EOLC communications for pediatric patients should be further evaluated.

Multiple-organ effect of normobaric hyperoxia in neonatal rats.

PURPOSE: Prolonged exposure to normobaric hyperoxia (NH) is associated with blood leukocyte activation and sequestration in the lung. Whether NH-induced leukocyte activation and sequestration can affect extrapulmonary organs or blood cellular profile has not been systematically investigated. We studied simultaneous changes in blood cellular profile and pulmonary, renal, and intestinal histology during NH and after return to air breathing ("weaning"). MATERIALS AND METHODS: One-day-old rats were exposed to 2 to 4 days of NH (FiO2 >0.98) or normoxia (FiO2 = 0.21), with or without weaning. Pups were then euthanized and 100 microL of blood was collected (cardiac puncture) for differential white blood cells analysis (n = 12 per group). The lungs, a piece of distal ileum, and the left kidney were removed for histologic evaluation. RESULTS: Both NH and weaning generated significant increases in blood neutrophil count, whereas lymphocyte population was significantly increased only after weaning (P < .05; analysis of variance with Bonferroni correction for multiple comparisons). Normobaric hyperoxia created mild increases in the renal tubular necrosis, dilation, regeneration, and interstitial inflammation. A significant increase in the intestinal serosal and submucosal vasodialation and vascularization occurred 1 day after weaning from 4 days of NH (P < .001). These extrapulmonary events coincided with the development of histologic manifestations of pulmonary oxygen toxicity. CONCLUSIONS: Development of pulmonary oxygen toxicity in neonatal rats is associated with significant changes in differential leukocyte counts and histologic alterations in the kidney and ileum. We speculate that activation of circulating leukocytes and/or direct effect of NH may affect certain peripheral organs independently from the NH-induced pulmonary pathology.

Is the Australian hospital system adequately prepared for terrorism?

Australian hospitals need to be prepared to deal with mass casualties from terrorist strikes, including bomb blasts and chemical, biological and radiation injury. Injuries from bomb explosions are more severe than those commonly seen in Australian hospitals. In disasters involving mass casualties in urban areas, many of the injured make their own way to hospital, often arriving before the more seriously injured casualties. Major hospitals in Australia should plan for large numbers of undifferentiated and potentially contaminated casualties arriving with minimal warning. It is critical that experienced and trained senior medical officers perform the triage of casualties in emergency departments, with frequent reassessment to detect missed injuries (especially pulmonary blast injury). Hospitals require well developed standard operating procedures for mass casualty events, reinforced by regular drills. Preparing for a major event includes training staff in major incident management, setting up an operational/control unit, nominating key personnel, ensuring there is an efficient intra-hospital communication system, and enhancing links with other emergency services and hospitals.

Staff exposure to ionizing radiation in a major trauma centre.

BACKGROUND: The purpose of the present paper was to determine the safety of staff members with regard to ionizing radiation in a major trauma centre in a 19-month period. METHODS AND RESULTS: A group of five doctors, five nurses and a trauma orderly wore personal radiation monitors under lead aprons while at work. The highest individual cumulative result after 586 days was 0.18 mSv for a nurse. If the exposure rate to ionizing radiation was constant, this would be equivalent to 0.114 mSv per year. Therefore the results are well below the recommended occupational dose limit of 20 mSv per year. CONCLUSION: Wearing of lead aprons during trauma resuscitation appears to be safe and provides adequate protection.