Serratiopeptidase reduces the invasion of osteoblasts by Staphylococcus aureus

Finding new strategies to counteract periprosthetic infection and implant failure is a main target in orthopedics. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter and kill osteoblasts, to stimulate pro-inflammatory chemokine secretion, to recruit osteoclasts, and to cause inflammatory osteolysis. Moreover, by entering eukaryotic cells, staphylococci hide from the host immune defenses and shelter from the extracellular antibiotics. Thus, infection persists, inflammation thrives, and a highly destructive osteomyelitis occurs around the implant. The ability of serratiopeptidase (SPEP), a metalloprotease by Serratia marcescens, to control S. aureus invasion of osteoblastic MG-63 cells and pro-inflammatory chemokine MCP-1 secretion was evaluated. Human osteoblast cells were infected with staphylococcal strains in the presence and in the absence of SPEP. Cell proliferation and cell viability were also evaluated. The release of pro-inflammatory chemokine MCP-1 was evaluated after the exposure of the osteoblast cells to staphylococcal strains. The significance of the differences in the results of each test and the relative control values was determined with Student’s t-test. SPEP impairs their invasiveness into osteoblasts, without affecting the viability and proliferation of bone cells, and tones down their production of MCP-1. We recognize SPEP as a potential tool against S. aureus bone infection and destruction

The use of an aircraft test stand for VTOL handling qualities studies

The VTOL flight tests stand for testing control concepts on the X-14B VSS aircraft in hover, is described. This stand permits realistic and safe piloted evaluation and checkout of various control systems and of parameter variations within each system to determine acceptability to the pilot. Pilots can use it as a practical training tool to practice procedures and flying techniques and become familiar with the aircraft characteristics. Some examples of test experience are given. The test stand allows the X14B to maneuver in hover from centered position + or - 9.7 deg in roll and + or - 9.3 deg in pitch, about + or - 6 deg in yaw, and + or - 15 cm in vertical translation. The unique vertical free flight freedom enables study of liftoffs and landings with power conditions duplicated. The response on the stand agrees well with that measured in free hovering flight, and pilot comments confirm this

Studi Anatomi Intestinum Krasum pada Kalong Kapauk (Pteropus Vampyrus)

Pteropus vampyrus is one of the animal species that plays an important role in the ecosystem by spreading the seeds of the edible fruits. Feed variations on each animal greatly affect the digestive tract. This study was aimed to determine the anatomical structure of the large intestine. Five adult large flying foxes weighing 500-800 g were used as the sample in this study. Large intestines were embedded in paraffin and stained with H & E method. H & E staining resulted in the presence of intestinal villi on colon and rectum

Comparison of the action of different proteases on virulence properties related to the staphylococcal surface.

AIMS: The purpose of this study was to evaluate the antimicrobial efficacy of five different proteases belonging to two different families on Staphylococcus aureus and Staphylococcus epidermidis strains. METHODS AND RESULTS: We used three serine proteases and two metalloproteases in single species biofilm formation assays and in human cell invasion processes. Following each protease incubation with bacterial cells, surface protein patterns were analysed by SDS-PAGE and zymography. Some differently expressed proteins were identified by mass spectrometry. CONCLUSIONS: The effect of tested proteases on biofilm formation was not related to the protease category but was strain-dependent and was related to the biofilm formation capacity of each staphylococcal strain. Some proteases showed a nonspecific and indiscriminate effect on surface proteins, while others induced a discrete and reproducible action on protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of the surface-related virulence factors is a promising avenue to overcome persistent infections caused by bacterial biofilms. To this end, we show here that proteases, in particular the metalloprotease serratiopeptidase, can interfere with adhesion and invasion of eukaryotic cells and biofilm formation in staphylococci and their use could represent a viable treatment for the development of novel combination therapie

Carbapenem-resistant Pseudomonas aeruginosa with acquired bla(vim) metallo-beta-lactamase determinants, Italy.

6nonenoneROSSOLINI G.M.; RICCIO M.L.; CORNAGLIA G.; PAGANI L.; LAGATOLLA C.; SELAN L. AND FONTANA R.Rossolini, G. M.; Riccio, M. L.; Cornaglia, G.; Pagani, L.; Lagatolla, Cristina; Selan, L. AND FONTANA R

PENGARUH KUALITAS LAYANAN TERHADAP KEPUASAN PELANGGAN (Studi Pada Hotel Timor Megah Kota SoE, TTS)

More and more manufacturers are involved in meeting the needs and desires of customers, causing each company to set orientation to customer satisfaction as the main goal. There is a tendency that customer satisfaction or dissatisfaction can be influenced by the quality of services provided by the company. This research was conducted at the Timor Megah Hotel Kota SoE, TTS Regency. The purpose of this study is to determine the effect of service quality that can be measured by several dimensions, including: tangibles, responsiveness, reliability, assurance, and empathy on customer satisfaction Timor Megah Hotel. The independent variable (X) of this study is the dimension of service quality consisting of tangibles (X1), responsiveness (X2), reliability (X3), assurance (X4), and empathy (X5). While the dependent variable (Y) of this study is customer satisfaction. The sampling technique used is the Accidental Sampling technique, with a total sample of 99 people with the criteria of having stayed more than twice and staying period for 4 years (2015-2018). Data collection techniques used were interviews, questionnaires and literature study. After the data is collected, the data is then analyzed using descriptive techniques and multiple linear regression. From the analysis it is known that tangibles, responsiveness, reliability, assurance and empathy simultaneously and partially have a significant effect on customer satisfaction. From the results of the analysis can also be made mathematical equations: Y = 3,568 + 0,150X1 + 0,245X2 + 0,209X3 + 0,192X4 + 0,362X5. The coefficient of determination obtained shows that 87.2% of the level of customer satisfaction is influenced by tangibles, responsiveness, reliability, assurance and empathy.   Keywords: Tangibles, Responsiveness, Reliability, Assurance, Empathy and Customer Satisfaction

Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm

Background The use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases. Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown. Methods Spep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system. Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses. The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method. Results In this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity. Conclusions New knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential “antipathogenic agent” capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity

A new anti-infective strategy to reduce the spreading of antibiotic resistance by the action on adhesion-mediated virulence factors in Staphylococcus aureus.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. S. aureus expresses a wide array of secreted and cell surface-associated virulence factors, including proteins that promote adhesion to damaged tissue and to the surface of host cells, and that bind proteins in blood to help evade immune responses. Furthermore, surface proteins have a fundamental role in virulence related properties of S. aureus, including biofilm formation. The present study evaluates the anti-infective capabilities of a secreted protein of Serratia marcescens (serratiopeptidase, SPEP), in impairing some staphylococcal virulence-related properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. It is not assessed if this action is due to the proteolytic activity of SPEP or to a specific mechanism which triggers an out/inside signal. Proteomic studies performed on surface proteins extracted from SPEP treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, SdrD, Sbi, EF-Tu and EF-G. EF-Tu and EF-G are known to perform a variety of function, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential "anti-infective agent" capable to hinder the entry of S. aureus into human tissues, and also impairs the ability of this pathogen to adhere to prostheses, catheters and medical device

PCS-15 Study Anatomy of Testis and Epididymis of Timorese Fruit Bats (Pteropus vampyrus)

Fruit bats (Pteropus vampyrus) is one of the bats in Timor Island, East Nusa Tenggara (NTT). Fruit bats have unique behaviour when mating which is hanging upside down (Corbet and Hill, 1992). The ability of male bat in maintaining the position when mating is according to the characteristics of male fruit bat reproduction system morphology. Testes are male genital organs which synthesis androgen hormone (especially testosterone) and the site where spermatogenesis takes place and eventually  producing sperm. Epididymis plays role in transporting, storaging and maturating the sperm (Syahrum et al., 1994)