The effect of combining bone morphogenetic proteins -2 and -6 on osteoblastic differentiation and bone

Bone morphogenetic proteins-2 and -7 (BMP-2 and -7) are the only two members of the BMP subfamily approved to date to be used in combination with collagen type I in orthopedic surgery, although other BMPs have proven to also be highly osteoinductive. All the osteogenic BMPs signal through Smad-1/-5/-8 phosphorylation, but they have different preferences for the BMP receptors they use. Very high supraphysiological doses of BMPs have been used in the clinics for the treatment of non-union fractures and spinal fusions. Besides the high cost of these treatments, safety concerns have been recently raised. Hence there is an active field in finding alternatives to the most classical collagen + BMP-2 system. The aim of this work was to study the effect of combining two osteogenic BMPs (-2 and -6) belonging to different groups within the subfamily, and with different affinities to the existing BMP receptors. Both the growth and osteoblastic differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs) under these conditions were studied, as well as in vivo ectopic bone formation when the BMPs were combined with collagen type I sponges. We show that the effect of these two growth factors is additive and that their combination might be helpful to accelerate in vivo osteogenesis while reducing the amount of each individual BMP used.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Enterococcus faecalis inhibits Klebsiella pneumoniae growth in polymicrobial biofilms in a glucose-enriched medium

Catheter-related urinary tract infections are one of the most common biofilm-associated diseases. Within biofilms, bacteria cooperate, compete, or have neutral interactions. This study aimed to investigate the interactions in polymicrobial biofilms of Klebsiella pneumoniae and Enterococcus faecalis, two of the most common uropathogens. Although K. pneumoniae was the most adherent strain, it could not maintain dominance in the polymicrobial biofilm due to the lactic acid produced by E. faecalis in a glucose-enriched medium. This result was supported by the use of E. faecalis V583 ldh-1/ldh-2 double mutant (non-producer of lactic acid), which did not inhibit the growth of K. pneumoniae. Lyophilized cell-free supernatants obtained from E. faecalis biofilms also showed antimicrobial/anti-biofilm activity against K. pneumoniae. Conversely, there were no significant differences in planktonic polymicrobial cultures. In summary, E. faecalis modifies the pH by lactic acid production in polymicrobial biofilms, which impairs the growth of K. pneumoniae

A model of maxilla resection to test new hybrid implants:macroporous titanium and tissue engineering elements

Maxillary bone loss in commonly found in humans, due to bone ageing, tooth loos, periodontal disease and, more severely, to trauma, radiotherapy and tumor resection. Masillofacial reconstructive surgery is a still unmet clinical demand, available therapies include grafting of autologous or heterologous bone tissue and/or the implantation of metallic plates, buy these treatments are still unable to resume form and function. The emrgence of 3D-printing technology applied to metal alloys now allows the manufacturing of customized, patient-tailored prosthetic implants. However, poor bone quiality at the implant site due to ageing or disease still hamper proper osseointegration. By combining Electron Beam Melting metal sintering and tissue engineering, we are developing hybrid maxillofacial implants, wher a metal framework of Ti6Al4V alloy confers both and appropiaate shape and mechanical stabilty, while stem cells and osteogenic molecules stimulate bone growth into the metal framework, thus pormoting osseointegration. We hereby present the in vitro work driving to the development of our hybrid maxillofacial prostheses, as well as the setting up of an in vivo model of complete maxilla full resection, created in order to test the prostheses in a preclinical studyUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Bone marrow mesenchymal stem cells, collagen scaffold and BMP-2 for rat spinal fusio

The use of autograft for posterolateral spinal fusion, continue being considered the gold standard for the treatment of spine pathologies. However, due to complications such as donor site morbidity, increased operating time, and limited supply, the use of allograft has become an acceptable practice especially in multisegment arthrodesis or in patients with previous graft harvests. Since their use involves the risk of immune response or disease transmission and fusion rates are not as good as with autogenous bone, a variety of bone graft substitutes are being studied to obtain a better alternative. Osteoinductive growth factors, which initiate the molecular cascade of bone formation and play a key role in the development and regeneration of the skeletal system, have been shown to be effective in numerous animal studies. These molecules must be used in combination with a biomaterial to avoid their dispersion from the application site. On the other hand, it is well known that cultured bone marrow cells, harvested from adult bone marrow, may contribute to the regeneration of bone. Thus, hybrid constructs can be used as alternatives to autologous and allogenic grafts. In this study, we have evaluated different combination of cultured bone marrow cells with recombinant human osteoinductive growth factors, all of them in combination with a natural polimeric carrier, for the promotion of posterolateral spinal fusion in rats. Supported by grants from the Red de Terapia Celular (RD12/0019/0032), Spanish Government BIO2012-34960, and the Andalusian Government (P11-CVI 07245).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A synthetic collagen-binding arg-gly-asp (rgd) biomimetic peptide enhances bone cell differentiation

RGD (arginine-glycine-aspartic acid) peptides have shown some promising abilities to promote the attachment of cells to biomaterials and to direct their differentiation. However, anchoring these peptides to the biomaterial’s surface is mandatory and usually implies several chemical linking steps. The aim of this work was to design and characterize a synthetic RGD biomimetic peptide that includes a collagen-binding domain for easy one-step functionalization of absorbable collagen sponges (ACSs), which are of frequent use in orthopaedic surgery. The stable binding of biotinylated CBD-RGD peptide loaded onto ACSs was confirmed using chemiluminisence detection after washing of the sponges. Furthermore, the effect of the peptide on MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs) in vitro was characterized in terms of caspase activity, proliferation, alkaline phosphatase (ALP) activity, matrix mineralization and formation of focal adhesions. Finally, a rat ectopic osteogenesis model was used to determine if the co-administration of CBD-RGD could lower the dose of BMP-2 necessary to induce bone formation. The CBD-RGD peptide was demonstrated to bind stable to ACSs, even after extensive washing. In vitro, the peptide did not induce apoptosis of the cells, but positively affected both cell growth and differentiation. It also seemed to affect the cytoskeleton arrangement of MC3T3-E1 cells, favoring the establishment of focal adhesions. At last, the in vivo experiments showed that ACSs functionalized with this peptide and loaded with a subfunctional dose of BMP-2 gave rise to ectopic bone. In conclusion. the combination of CBD-RGD with the currently used collagen/BMP system might be a promising approach to improve osteogenesis and to reduce the doses of BMPs needed in clinical orthopaedics.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A C˄S-cyclometallated gold(III) complex as novel antibacterial candidate against drug-resistant bacteria

The worldwide emergence and spread of infections caused by multidrug-resistant bacteria endangers the efficacy of current antibiotics in the clinical setting. The lack of new antibiotics in the pipeline points to the need of developing new strategies. Recently, gold-based drugs are being repurposed for antibacterial applications. Among them, gold(III) complexes have received increasing attention as metal-based anticancer agents. However, reports on their antibacterial activity are scarce due to stability issues. The present work demonstrates the antibacterial activity of the gold(III) complex 2 stabilized as C∧S-cycloaurated containing a diphenylphosphinothioic amide moiety, showing minimum inhibitory concentration (MIC) values that ranged from 4 to 8 and from 16 to 32 mg/L among Gram-positive and Gram-negative multidrug-resistant (MDR) pathogens, respectively. Complex 2 has a biofilm inhibitory activity of only two to four times than its MIC. We also describe for the first time a potent antibacterial synergistic effect of a gold(III) complex combined with colistin, showing a bactericidal effect in less than 2 h; confirming the role of the outer membrane as a permeability barrier. Complex 2 shows a low rate of internalization in Staphylococcus aureus and Acinetobacter baumannii; it does not interact with replication enzymes or efflux pumps, causes ultrastructural damages in both membrane and cytoplasmic levels, and permeabilizes the bacterial membrane. Unlike control antibiotics, complex 2 did not generate resistant mutants in 30-day sequential cultures. We detected lower cytotoxicity in a non-tumoral THLE-2 cell line (IC50 = 25.5 μM) and no acute toxicity signs in vivo after an i.v. 1-mg/kg dose. The characterization presented here reassures the potential of complex 2 as a new chemical class of antimicrobial agents

Long-term programme of biophysical monitoring of the personnel involved in the construction of the new safe confinement

The international Shelter Implementation Plan (SIP) foresees a step-by-step transformation of the Chornobyl site into the safe condition. The New Safe Confinement (NSC), is an unprecedented 108-meter tall structure over the destroyed Unit 4 of the Chernobyl Nuclear Power Plant, and is a central element of SIP. Many workers involved in SIP may have a direct contact with fragments of the irradiated fuel, fuel-containing aerosols, and other contaminated material at the Chornobyl industrial site. Isotopes of plutonium, americium, strontium, and cesium are dominating in internal exposure of SIP workers. The safety of workers is a high priority of SIP. Starting from 2004 the Ukrainian Radiation Protection Institute (RPI) is performing a large-scale Internal Dosimetry Program as an integral part of SIP. Pu contents in fecal and urine samples and the whole-body counters‟ (WBC) data and are the main source of the quantitative data used for the dose assessment. The RPI radiochemical laboratories in Kyiv and at the Chornobyl site employed the standard radiochemical technique and equipped with ninety six alpha-spectrometers. The range of WBCs includes the scanning low-background WBC, four Canberra FastScan WBCs, and four chair-type WBCs. The car-borne WBC is located in Kiev and reserved for the emergency purposes. As on September 2017 the Internal Dosimetry Program has covered more than 17 000 workers, which undergone 1 230 000 measurements of ¹³⁷Cs on WBCs, 87 000 measurements of ²³⁹⁺²⁴⁰Pu contents in fecal samples and 4 400 measurements of ²³⁹⁺²⁴⁰Pu contents in urine samples. Such a large-scale programme ensured a reliable monitoring of intakes of the insoluble radioactive material.У міжнародному Плані здійснення заходів (ПЗЗ) передбачається поетапне перетворення чорнобильського майданчика у безпечний стан. Новий безпечний конфайнмент є безпрецедентною структурою заввишки 108 м над зруйнованим 4-м блоком ЧАЕС, він є центральним елементом ПЗЗ. Багато працівників, залучених у ПЗЗ, можуть мати прямий контакт із фрагментами опроміненого палива, паливовмісними аерозолями та іншими забрудненими матеріалами на промисловому майданчику в Чорнобилі. Ізотопи плутонію, америцію, стронцію і цезію домінують у внутрішньому опроміненні працівників ПЗЗ. Безпека працівників має високий пріоритет у ПЗЗ. Починаючи з 2004 р., Науково-дослідний інститут радіаційного захисту АТН України (ІРЗ) здійснює великомасштабну програму дозиметрії внутрішнього опромінення як невід‟ємну частину ПЗЗ. Дані вмісту плутонію у пробах калу і сечі та лічильників випромінювань людини (ЛВЛ) і основним джерелом кількісних даних, що використовуються для оцінки дози. Радіохімічні лабораторії ІРЗ у Києві і на майданчику в Чорнобилі використовували стандартну радіохімічну методику, вони оснащені 96 альфа-спектрометрами. Номенклатура ЛВЛ включає в себе скануючий низькофоновий ЛВЛ, чотири ЛВЛ Canberra FastScan і чотири ЛВЛ типу “крісло”. Мобільний ЛВЛ розташований у Києві і зарезервований для надзвичайних цілей. Станом на вересень 2017 р. рамками програми дозиметрії внутрішнього опромінення було охоплено понад 17 000 працівників, які пройшли 1 230 000 вимірювань ¹³⁷Cs на ЛВЛ, 87 000 вимірювань вмісту ²³⁹⁺²⁴⁰Pu у пробах калу і 4 400 вимірювань вмісту ²³⁹⁺²⁴⁰Pu у пробах сечі. Така великомасштабна програма забезпечила надійний контроль надходжень нерозчинного радіоактивного матеріалу.В международном Плане осуществления мероприятий (ПОМ) предусматривается поэтапное преобразование чернобыльской площадки в безопасное состояние. Новый безопасный конфайнмент – это беспрецедентная структура высотой 108 м над разрушенным 4-м блоком ЧАЭС, он является центральным элементом ПОМ. Многие работники, вовлеченные в ПОМ, могут иметь прямой контакт с фрагментами облученного топлива, топливосодержащими аэрозолями и другими загрязненными материалами на промышленной площадке в Чернобыле. Изотопы плутония, америция, стронция и цезия доминируют во внутреннем облучении работников ПОМ. Безопасность работников имеет высокий приоритет в ПОМ. Начиная с 2004 г., Научно-исследовательский институт радиационной защиты АТН Украины (ИРЗ) осуществляет крупномасштабную программу дозиметрии внутреннего облучения как неотъемлемую часть ПОМ. Данные содержания плутония в пробах кала и мочи, а также счетчиков излучений человека (СИЧ) являются основным источником количественных данных, используемых для оценки дозы. Радиохимические лаборатории ИРЗ в Киеве и на площадке в Чернобыле использовали стандартную радиохимическую методику, они оснащены 96 альфа-спектрометрами. Номенклатура СИЧ включает в себя сканирующий низкофоновый СИЧ, четыре СИЧ Canberra FastScan и четыре СИЧ типа “кресло”. Мобильный СИЧ расположен в Киеве и зарезервирован для чрезвычайных целей. По состоянию на сентябрь 2017 г. в рамках программы дозиметрии внутреннего облучения было охвачено более 17 000 работников, которые прошли 1 230 000 измерений ¹³⁷Cs на СИЧ, 87 000 измерений содержания ²³⁹⁺²⁴⁰Pu в пробах кала и 4 400 измерений содержания ²³⁹⁺²⁴⁰Pu в пробах мочи. Такая крупномасштабная программа обеспечила надежный контроль поступлений нерастворимого радиоактивного материала

Некоторые подходы к совершенствованию регионально-институциональной основы курортно-гостиничных услуг в Автономной республике Крым

Рассмотрены подходы (институциональный, региональный, проблемно-ориентированный и маркетинговый) к разработке регионально-институциональной модели курортно-гостиничного хозяйства как одной из важнейших составляющих институциональной модели курортно-рекреационного комплекса Автономной Республики Крым.Розглядаються підходи (інституційний, регіональний, проблемно-орієнтований і маркетинговий) до розробки регіонально-інституційної моделі курортно-готельного господарства як однієї з найважливіших складових інституційної моделі курортно-рекреаційного комплексу Автономної Республіки Крим

Coronavirus Papain-like Proteases Negatively Regulate Antiviral Innate Immune Response through Disruption of STING-Mediated Signaling

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction

Novel gold(III)-dithiocarbamate complex targeting bacterial thioredoxin reductase: antimicrobial activity, synergy, toxicity, and mechanistic insights

IntroductionAntimicrobial resistance is a pressing global concern that has led to the search for new antibacterial agents with novel targets or non-traditional approaches. Recently, organogold compounds have emerged as a promising class of antibacterial agents. In this study, we present and characterize a (C^S)-cyclometallated Au(III) dithiocarbamate complex as a potential drug candidate.Methods and resultsThe Au(III) complex was found to be stable in the presence of effective biological reductants, and showed potent antibacterial and antibiofilm activity against a wide range of multidrug-resistant strains, particularly gram-positive strains, and gram-negative strains when used in combination with a permeabilizing antibiotic. No resistant mutants were detected after exposing bacterial cultures to strong selective pressure, indicating that the complex may have a low propensity for resistance development. Mechanistic studies indicate that the Au(III) complex exerts its antibacterial activity through a multimodal mechanism of action. Ultrastructural membrane damage and rapid bacterial uptake suggest direct interactions with the bacterial membrane, while transcriptomic analysis identified altered pathways related to energy metabolism and membrane stability including enzymes of the TCA cycle and fatty acid biosynthesis. Enzymatic studies further revealed a strong reversible inhibition of the bacterial thioredoxin reductase. Importantly, the Au(III) complex demonstrated low cytotoxicity at therapeutic concentrations in mammalian cell lines, and showed no acute in vivo toxicity in mice at the doses tested, with no signs of organ toxicity.DiscussionOverall, these findings highlight the potential of the Au(III)-dithiocarbamate scaffold as a basis for developing novel antimicrobial agents, given its potent antibacterial activity, synergy, redox stability, inability to produce resistant mutants, low toxicity to mammalian cells both in vitro and in vivo, and non-conventional mechanism of action