Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Skeletal evidence for violent trauma from the bronze age Qijia culture (2,300-1,500 BCE), Gansu Province, China.

This research explores how social and environmental factors may have contributed to conflict during the early Bronze Age in Northwest China by analyzing violent trauma on human skeletal remains from a cemetery of the Qijia culture (2300-1500 BCE). The Qijia culture existed during a period of dramatic social, technological, and environmental change, though minimal research has been conducted on how these factors may have contributed to violence within the area of the Qijia and other contemporaneous material cultures. An osteological assessment was conducted on 361 individuals (n = 241 adults, n = 120 non-adults) that were excavated from the Mogou site, Lintan County, Gansu, China. Injuries indicative of violence, including sharp- and blunt-force trauma that was sustained ante- or peri-mortem, were identified, and the patterns of trauma were analysed. Violent injuries were found on 8.58% (n = 31/361) of individuals, primarily adult males. No evidence of trauma was found on infants or children. Cranial trauma was found on 11.8% (n = 23/195) of the adult individuals examined. Of these, 43.5% (n = 10/23) presented with severe peri-mortem craniofacial trauma. The high rate of perimortem injuries and their locations indicate lethal intent. This lethality, in addition to the fact that individuals with trauma were predominantly male, suggest intergroup violence such as raiding, warfare, or feuding. Both social and environmental factors may have contributed to this conflict in the TaoRiver Valley, though future systematic archaeological and paleoenvironmental data will be needed to disentangle the many potential causal factors.

Anti-inflammatory and antioxidant effect of melatonin on recovery from muscular trauma induced in rats.

In recent decades, the number of people who practice sports has grown exponentially, increasing the number of muscular injuries. Trauma injury occurs when the muscle is exposed to a sudden compression force. Melatonin (MLT) has often been cited in the literature as a potent antioxidant and anti-inflammatory agent. This study was designed to evaluate MLT action on muscle tissue in Wistar rats in an experimental model of muscle trauma. Twenty-eight Wistar rats were used, divided into four groups: CO (Control), CO + MLT (Control + Melatonin), T (Trauma) and T + MLT (Trauma + Melatonin). MLT (20 mg/kg) was administered (ip) daily at dusk until day 7. The trauma occurred on day 1, 2 h before the first MLT application. On day 8, muscle tissue was collected for histological analysis (HE), immunohistochemistry (TNF-α and NFκB), evaluation of oxidative stress through analysis of lipoperoxidation by TBARS and activity of SOD and GPx enzymes, and analysis of nitrites and nitrates. In the evaluation of TBARS and SOD, we observed a significant increase in the T group and a significant decrease in the T + MLT group. In the evaluation of GPx, there was a significant increase in the T group and a significant decrease in the T + MLT group. The histological analysis of muscle tissue revealed structural changes of muscle fibers and inflammatory infiltrate in the T group but a decrease in this damage in the T + MLT group. In the immunohistochemical evaluation, increased expression of TNFα and NFκB proteins in the T group was observed and a significant decrease of this expression in the T + MLT group. MLT was shown to attenuate oxidative damage and to diminish the expression of inflammatory proteins and tissue damage in this experimental model.

Prehistoric massacre revealed. Perimortem cranial trauma from Potocani, Croatia.

ABSTRACT: During archaeological rescue excavations carried out in 2007 at Potocani in continental Croatia, a pit containing numerous human skeletal remains (MNI = 41) was discovered. The remains were mostly articulated but also commingled and showed no clear pattern of organization. There were no associated artifacts, just a few pottery fragments probably belonging to the Copper Age Lasinja Culture (c. 4300 to 3950 BCE). Anthropological analyses suggest the presence of individuals of all ages and both sexes with many crania exhibiting various perimortem injuries. Three human bone samples from different layers were dated to around 4100 cal BCE by radiocarbon analysis. These radiocarbon dates combined with other aspects of archaeological context, indicate that the deposition was a single episode rather than a long-term accumulation. All this suggests a single violent encounter (massacre). Here we present results of the bioarchaeological analysis of four adult crania with clear signs of perimortem trauma. These include blunt force trauma as well as cuts and penetrating injuries indicating the use of different weapons/tools.

Enhanced Cutaneous Wound Healing In Vivo by Standardized Crude Extract of Poincianella pluviosa.

Wound healing is a complex process that involves several biological events, and a delay in this process may cause economic and social problems for the patient. The search continues for new alternative treatments to aid healing, including the use of herbal medicines. Members of the genus Caesalpinia are used in traditional medicine to treat wounds. The related species Poincianella pluviosa (DC.) L.P. Queiroz increases the cell viability of keratinocytes and fibroblasts and stimulates the proliferation of keratinocytes in vitro. The crude extract (CE) from bark of P. pluviosa was evaluated in the wound-healing process in vivo, to validate the traditional use and the in vitro activity. Standardized CE was incorporated into a gel and applied on cutaneous wounds (TCEG) and compared with the formulation without CE (Control) for 4, 7, 10, or 14 days of treatment. The effects of the CE on wound re-epithelialization; cell proliferation; permeation, using photoacoustic spectroscopy (PAS); and proteins, including vascular endothelial growth factor (VEGF), superoxide dismutase 2 (SOD-2) and cyclooxygenase 2 (COX-2) were evaluated. The TCEG stimulated the migration of keratinocytes at day 4 and proliferation on the following days, with a high concentration of cells in metaphase at 7 days. Type I collagen formed more rapidly in the TCEG. PAS showed that the CE had permeated through the skin. TCEG stimulated VEGF at day 4 and SOD-2 and COX-2 at day 7. The results suggest that the CE promoted the regulation of proteins and helped to accelerate the processes involved in healing, promoting early angiogenesis. This led to an increase in the re-epithelialized surface, with significant mitotic activity. Maturation of collagen fibers was also enhanced, which may affect the resistance of the extracellular matrix. PAS indicated a correlation between the rate of diffusion and biological events during the healing process. The CE from P. pluviosa appears promising as an aid in healing.

A cannabinoid type 2 receptor agonist attenuates blood-brain barrier damage and neurodegeneration in a murine model of traumatic brain injury.

After traumatic brain injury (TBI), inflammation participates in both the secondary injury cascades and the repair of the CNS, both of which are influenced by the endocannabinoid system. This study determined the effects of repeated treatment with a cannabinoid type 2 receptor (CB(2) R) agonist on blood-brain barrier integrity, neuronal degeneration, and behavioral outcome in mice with TBI. We also looked for the presence of a prolonged treatment effect on the macrophage/microglial response to injury. C57BL/6 mice underwent controlled cortical impact (CCI) and received repeated treatments with a CB(2) R agonist, 0-1966, or vehicle. After euthanasia at 6 hr or 1, 2, 3, or 7 days postinjury, brains were removed for histochemical analysis. Blood-brain barrier permeability changes were evaluated by using sodium fluorescein (NaF). Perilesional degenerating neurons, injury volumes, and macrophage/microglia cells were quantified by stereological methods. Rota-rod and open-field testing were performed to evaluate motor function and natural exploratory behavior in mice. 0-1966 Treatment resulted in a significant reduction in NaF uptake and number of degenerating neurons compared with the vehicle-treated group. 0-1966-Treated mice demonstrated improvement on rota-rod and open-field testing compared with vehicle-treated mice. These changes in CCI mice treated with 0-1966 were associated with a prolonged reduction in macrophage/microglia cell counts. In conclusion, repeated treatments with a CB(2) R agonist, 0-1966, result in attenuated blood-brain barrier disruption and neuronal degeneration. In addition, repeated treatment with 0-1966 shows prolonged treatment effects on behavior and the macrophage/microglia cell response over several days.

When backyard fun turns to trauma: risk assessment of blunt ballistic impact trauma due to potato cannons.

Although potato cannons are an area of great interest among internet users, they are almost completely unknown in the medical community. These simple ballistic devices are made from plastic plumbing pipes and are powered with propellant gas from aerosol cans. By combustion of the gas-oxygen mixture, a high pressure is produced which propels the potato chunks through the barrel. It is the aim of this study to investigate the hazardous potential of these shooting devices. Test shots were performed using three illegally manufactured potato cannons that were confiscated by police authorities. Velocity, impulse, kinetic energy, and energy density were calculated. The risk of head and chest injuries was investigated by using Sturdivan's Blunt Criterion (BC), an energy based five parametric trauma model assessing the vulnerability to blunt weapons, projectile impacts, and behind-body-armor exposures. The probability of lethality due to blunt impact trauma to the chest was assessed using Sturdivan's lethality model. For potential head impacts, all test shots far exceeded the critical BC (head) value which corresponds to a 50% risk of skull fracture. The risk of injury with regard to chest impacts was similar. All but two test shots far exceeded the critical BC (chest) value corresponding to a 50% risk of sustaining a thoracic skeletal injury of Abbreviated Injury Scale 2 or 3. The probability of a lethal injury due to blunt chest impact was as high as 20%. To conclude, this work demonstrates that potato cannons should be considered dangerous weapons rather than as toys used by adventurous adolescents.

Contrast-enhanced ultrasound for blunt hepatic trauma: an animal experiment.

OBJECTIVE: The aim of this study was to study the value of contrast-enhanced ultrasound (CEUS) in the diagnosis of active hemorrhage and intraparenchymal lesions in blunt hepatic trauma versus conventional ultrasound (US). METHODS: Twenty heparinized and anesthetized domestic pigs have been created to animal models with blunt hepatic trauma by a special impacting device. Conventional US and CEUS were performed to determine if hepatic traumas were present. RESULTS: Active hemorrhage, the presence of intraparenchymal lesions, and sonographic pattern were evaluated for conventional US and CEUS, as compared with laparotomy and pathologic findings. Contrast-enhanced US detected active hemorrhage from the injured livers in all cases, but conventional US did not find that in any case. The sensitivity of CEUS and conventional US in diagnosing intraparenchymal lesions of blunt hepatic trauma were 100% and 60%, respectively. CONCLUSIONS: Contrast-enhanced US is more sensitive than conventional US in determining the active hemorrhage and intraparenchymal lesions in blunt hepatic trauma.

Trauma-induced changes in diaphyseal cross-sectional geometry in two elites from Copan, Honduras.

This research utilized biplanar radiographs to estimate cross-sectional biomechanical properties for the skeletal remains of two elite individuals from the Early Classic period (ca. AD 400-600) of Copan, Honduras: K'inich Yax K'uk' Mo' (Hunal Burial 95-2), founder of the Early Classic Dynasty at Copan, and the primary female interment (Burial 37-8) from the Motmot tomb. Both individuals survived severe blunt-force insults to the right forearm. Gross skeletal examination and evaluation of the radiographs for K'inich Yax K'uk' Mo' suggest that these traumas resulted from, at least in part, disuse atrophy of the affected forearm skeletal elements. Gross and radiologic evaluation of the Motmot remains countered the possibility that she suffered from a metabolic bone disease, and confirmed the presence of a well-healed parry fracture of the right ulna. The degree of asymmetry in cross-sectional biomechanical properties reported here for K'inich Yax K'uk' Mo' is likely the secondary result of the described blunt-force trauma. The results obtained for the principal Motmot interment are not as dramatic, but suggest subtle changes to humeral cross-sectional geometry subsequent to trauma.

Potential of MRI and 31P MRS in the evaluation of experimental testicular trauma.

OBJECTIVES: To prognosticate and assess the metabolic status of germ cells of the testis after unilateral blunt testicular trauma using both magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). MRI is a noninvasive technique suitable for evaluating testicular trauma, and MRS is useful in assessing the metabolic status of the testis. METHODS: The right testis of 35 male prepubertal Wistar rats, aged 30 days, was explored through an inguinal incision. The rats were randomized into control (group 1, sham surgery, n = 10) and study (n = 25) groups. The study group received unilateral blunt testicular trauma to the right testis. T(1)- and T(2)-weighted proton MRI of the ipsilateral testis were taken 6 hours after sham surgery or injury, and the rats were stratified on the basis of the absence or presence of intratesticular hemorrhage on MRI into groups 2 (n = 14) and 3 (n = 11), respectively. At 60 days of age, the contralateral testis of each rat was evaluated by 31P MRS and histologic examination. Quantification of phosphomonoesters, phosphodiesters, phosphocreatine, and adenosine triphosphate (gamma, alpha, and beta) was done. RESULTS: A statistically significant difference (P <0.05) in the phosphomonoester/adenosine triphosphate ratio, seminiferous tubular diameter, and Johnsen score of the contralateral testis was observed, indicating decreased testicular maturation of the contralateral testis in group 3 rats compared with groups 1 and 2. CONCLUSIONS: MRI after testicular trauma helped to stratify the extent of injury as determined by the presence or absence of intratesticular hemorrhage with prognostic value; 31P MRS and histologic examination revealed that testicular trauma significantly affects the maturation of the contralateral testis.

Macrophage cyclooxygenase expression, immunosuppression, and cardiopulmonary dysfunction after blunt chest trauma.

BACKGROUND: Two series of experiments were performed in swine who received severe blunt chest trauma. The goals were to determine the time course of constitutive and inducible cyclooxygenase (COX) isozyme expression in pulmonary macrophages (Mphis), and to determine whether COX expression and cardiopulmonary dysfunction were altered when neutrophils (PMNs) were pharmacologically depleted with cyclophosphamide (CYC). METHODS: In series 1 (n = 17), anesthetized, mechanically ventilated swine were subjected to right chest trauma via captive bolt gun, hemorrhage, and a 60-minute shock period. In series 2 (n = 41), CYC (50 mg/kg intravenously) was administered 4 days before trauma, and the shock period was shortened to 30 minutes. In both series, hemodynamic support and supplemental oxygen were provided for an additional 60 to 90 minutes after shock. Mphis were isolated from serial bilateral bronchoalveolar lavages (BALs) and COX protein expression was measured with Western blots. RESULTS: In series 1, death occurred in 11 of 17. In survivors, Mphi COX-1 peaked at > 100 times baseline in both right BAL and left BAL by 60 minutes (before resuscitation). Changes in Mphi COX-2 were minimal. In series 2, before trauma, CYC (n = 16) reduced circulating and BAL PMNs by > 90% relative to control (n = 25, both p < 0.05) with no complicating side effects. After trauma, death occurred in 11 of 25 controls versus 9 of 16 with CYC. In survivors, PaO2/FIO2 was < 250 and PaCO2 was 25% higher on constant minute ventilation, indicating mismatched ventilation/perfusion; both changes were reduced with CYC (p < 0.05). In controls, bilateral histologic damage included edema, alveolar hemorrhage, and interstitial infiltrates. These changes were reduced by one third with CYC (p = 0.08). Trauma-induced changes in BAL protein, BAL elastase, or Mphi COX expression were not lessened by CYC. CONCLUSION: After unilateral chest trauma, Mphi COX-1, not COX-2, is induced bilaterally and before fluid resuscitation; CYC prevented PMN infiltration and attenuated structural and functional changes after resuscitation, which suggests that PMNs have a role in the pathogenic mechanism of secondary lung injury; Mphi COX expression and other injury markers were not altered by CYC; and since Mphis continued to express proinflammatory COX protein even after pretreatment with a powerful nonspecific immunosuppressant, and since there is residual alveolar capillary damage even in the absence of PMNs, it is logical to conclude that no single cell type or mediator is a practical therapeutic target and that novel resuscitation strategies must address multiple elements in the inflammatory cascade.

Importance of posttraumatic hypothermia and hyperthermia on the inflammatory response after fluid percussion brain injury: biochemical and immunocytochemical studies.

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30 degrees C) and hyperthermia (39 degrees C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37 degrees C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5 degrees C), hypothermia (30 degrees C), or hyperthermia (39 degrees C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within the anterior cortical segment (213.97 +/- 56.2 versus control 65.5 +/- 52.3 U/g of wet tissue; mean +/- SD) and posterior (injured) cortical and subcortical segments compared to sham-operated rats (305.76 +/- 27.8 and 258.67 +/- 101.4 U/g of wet tissue versus control 62.8 +/- 24.8 and 37.28 +/- 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region (P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment (P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values (P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 +/- 258.4 and 100.11 +/- 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome.

Cellular reactivity to mechanical axonal injury in an organotypic in vitro model of neurotrauma.

An in vitro model of traumatic brain injury is described that is based on organotypic cocultures (OTCs) of rat neocortex and thalamus connected by reciprocal axonal projections. Localized mechanical compression of this projection was inflicted with a mechanical device, and the effects on cell viability, axonal morphology, and protein expression levels were analyzed. Within 24 h after insult, major cell damage occurred in infragranular cortical layers containing the corticothalamic projection neurons and in thalamic regions adjacent to the mechanical impact as was assessed through the use of the vital stain Syto 21, and propidium iodide labeling. A small, but significant number of calretinin-positive interneurons in cortical and thalamic areas displayed symptoms of injury. Axonal elements, as revealed by neurofilament (NF-H/M) immunohistochemistry, in the corticothalamic transition zone displayed pathomorphological changes, such as axonal bulbs and swellings, already 4 h after insult. Densitometric analysis revealed that MAP-2a,b expression was not significantly changed within 4 h after injury. A significant reduction in MAP-2a,b amount was evident at 20 h after injury in thalamus (by 31.6%) and cortex (by 30%) maintained for 12 days in vitro (DIV), but not in OTCs aged 20 DIV. The axonally localized form MAP-2c significantly increased in cortex of 12-DIV OTCs at 4 and 20 h after insult (65.6% and 33.4%, respectively). MAP-2c levels in cortex of 20 DIV initially increased by 47.7% and declined below control values 20 h after injury. Thalamic areas revealed a delay in MAP-2c reactivity, in that expression was significantly elevated only at 20 h after injury (by 84.4% in 12-DIV and by 39.6% in 20-DIV OTCs, respectively). These data may reflect the regenerative ability of juvenile, but not of older neurons in response to mechanical axonal injury.

Histological evaluation of the canine decidual reactions induced by intraluminal injection of olive oil.

The present study deals with histological changes of the canine decidual reactions of the endometrium stimulated by intraluminal injection of olive oil with or without scratching injury using a wire. Olive oil caused conspicuous proliferation of the superficial endometrial glands. When olive oil injection was carried out after the scratching, cystic hyperplasia of basal glands as well as proliferation of superficial glands occurred. The results suggest that olive oil stimulates only proliferation of the superficial endometrial glands and the occlusion of the glandular orifices by some stimuli such as the traumatical injury would be needed to induce cystic hyperplasia of the basal glands.

[Immunomodulating action of myelopeptides in severe closed experimental craniocerebral injury in rats]. / Immunomoduliruiushchee deistvie mielopeptidov pri tiazheloi zakrytoi éksperimental'noi cherepno-mozgovoi travme u krys.

An immunocorrective effect of myelopeptides (MP) isolated from pig bone marrow cell culture supernatant in the early posttraumatic period in rats with severe cranial injury has been assessed. MP administration prevented cellular devastation of thymus and bone marrow, as well as spleen hyperplasia. The most marked MP effect was observed within the first 24 h after its administration. MP affected the functional and migration properties of both the entire population of lymphoid cells and individual subpopulations. MP had a pronounced protective effect against Staph. aureus infection during cranial trauma. MP completely prevented the death of animals and reduced more than 3-fold Staph. aureus persistence in the organism of animals. Anti-stress and protective effects of MP open vast prospects for their therapeutic and preventive application in the clinical practice.