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In recent years, researchers have shown an increased interest in using medicinal plants for the synthesis of silver nanoparticles (AgNPs) having various therapeutic properties. Centella asiatica (CA), a medicinal plant, has been used to treat minor burn wounds, psoriasis, and hypertrophic wounds among many other pathological conditions. The current study aimed to synthesize CA coated AgNPs (CA-AgNPs) with appropriate biocompatibility and various therapeutic properties, including antimicrobial and analgesic activities. The synthesized CA-AgNPs were characterized by ultraviolet-visible (UV-Vis) spectroscopy, zeta potential measurements, and fourier transform infrared (FT-IR) spectroscopy. The formation of spherical CA-AgNPs was confirmed by a single surface plasmon resonance (SPR) peak emerging at 420 nm wavelength by UV-Vis. The average hydrodynamic diameter and zeta potential of the particles were found to be 29.5 nm and -24.5 mV, respectively. The FT-IR analyses showed that the AgNPs were coated and stabilized by bioactive compounds from the CA extract. MTT cytotoxicity assay revealed that CA-AgNPs at ≤1 mM concentrations exhibited biocompatibility for L929 fibroblast cells. The antimicrobial activity of CA-AgNPs was confirmed by significant inhibition of Staphylococcus aureus and Escherichia coli. In addition, the analgesic effect of CA-AgNPs was investigated for the first time in the literature by tail-flick and hot plate methods, and statistically significant results were obtained for both methods. Taken together, these results suggest that CA-AgNPs can be used as an effective antibacterial and analgesic agent in a variety of biomedical applications, including coating wound dressings.

Objective: In this study, we aimed to present the pathogens detected as vaginal infection agents isolated from vaginal swab samples in adult patients admitted to the Department of Gynecology and Obstetrics of a private hospital in Istanbul for three years retrospectively.Material and Methods: Vaginal swabs of adult non-pregnant patients admitted to Gynecology and Obstetrics outpatient clinics of a private hospital in Istanbul between 2016-2018 were included in this study. The samples were transferred quickly to the laboratory after sampling, and the infectious agents were identified with conventional bacterial culture methods.Results: Identification results of 314 vaginal swab samples revealed that 28 (8.92%) had C. albicans, 13 (5.73%) had pathogens that cause aerobic vaginitis, and five (1.59%) had bacterial vaginosis agents. C. albicans was the highest among agents with a frequency of 8.92%, followed by E. coli (2.23%).Conclusion: Our retrospective analysis revealed that C. albicans is the most frequent pathogen causing vaginitis in our hospital. As the pathogens causing vaginitis vary from region to region, we believe that monitorization of the epidemiological data is important.

Objective: Raw milk is an important source of nutrients. Therefore, today, there is a great demand for raw milk consumption. The positive side of milk consumption on growth and development cannot be ignored, but unfortunately, pathogens in raw milk are always potential public health risks for transmission pathogens. Bacteria such as Enterobacteriaceae in normal flora can cause serious problems due to their extended-spectrum beta-lactamase (ESBL) production. These bacteria and their resistance genes have been reported in raw milk. In this matter, the aim of the study is to determine the status of blaCTX-M-1, blaCTX-M-2, blaTEM, and blaSHV genes responsible for the production of ESBL enzyme in Escherichia coli and Klebsiella spp. strains to identify risk factors in raw milk consumption and to gain an understanding of the epidemiology of these resistant strains. Materials and methods: A total of different 50 raw milk samples were collected and subjected to phenotypic microbiological analysis and Real-time PCR targeting blaCTX-M-1, blaCTX-M-2, blaTEM, and blaSHV genes. In the phenotypic analyses, suspicious isolates were identified by classical microbiological methods and antibiotic resistance profiles were revealed. Results: These results indicated that raw milk is a potential reservoir for ESBL producing E. Coli, Klebsiella spp. strains are obviously significant. And It was determined that CTX-M based ESBL genes are predominant in ESBL production. The present study revealed that raw milk is epidemiologically involved in the transmission of ESBL genes. Raw milk could be distributed to ESBL genes widely with which is consumed in Şanlı Urfa.

This review is prepared to give information about the molecular diagnosis methods of a new coronavirus SARS-CoV-2, which causes pandemic in a short time. In order to prevent the COVID-19 pandemic, which causes serious global destruction, the development of sensitive and specific laboratory diagnostic methods are crucial to identify cases quickly and reliably. Diagnostic tests have played an important role in the COVID-19 pandemic and will continue to play. Rapid sampling and testing of patients meeting the suspected case definition for COVID-19 is a priority for clinical management and outbreak control. The ability to detect SARS-CoV-2 in respiratory secretions is necessary to determine when an individual becomes infected and the potential for infecting other persons. Viral detection is used to identify individual patients, manage pandemics, as well as determine when and how quickly the virus is transmitted to a community. As communities seek to re-open after closure periods, the detection of both SARS-CoV-2 and specific antibodies that recognize the virus will become increasingly important as a tool for assessing infection and immunity in individuals and populations. For this purpose, nucleic acid amplification and serological tests are used to detect the SARS-CoV-2., Bu derleme, kısa bir süre içinde pandemiye sebep olan, yeni bir koronavirüs SARS-CoV-2’nin moleküler tanı yöntemleri ile ilgili bilgi vermek amacıyla hazırlanmıştır. Global olarak ciddi bir tahribata yol açan COVID-19 pandemisini önleyebilmek adına, hassas ve spesifik laboratuvar teşhis metotlarının geliştirilmesi, vakaların hızlı ve güvenilir şekilde tanımlanabilmesi için oldukça önemlidir. Diyagnostik testler COVID-19 pandemisinde önemli bir rol oynamıştır ve oynamaya devam edecektir. COVID-19 için şüphelenilen vaka tanımını karşılayan hastalardan hızla numune alınması ve test edilmesi klinik yönetim ve salgın kontrolü için bir önceliktir. Solunum sekresyonlarında SARS-CoV-2 saptama yeteneği, bireyin enfekte olma zamanını ve diğer kişilere bulaştırma potansiyelini belirlemek için gereklidir. Viral tespit, bireysel hastaların tanımlanması, pandemi yönetimi ve ayrıca virüsün bir topluluğa ne zaman bulaştığını ve ne kadar hızla yayıldığını belirlemek için kullanılır. Topluluklar, kapanma dönemlerinin ardından yeniden açılmaya çalıştıkça, hem SARS-CoV-2'nin hem de virüsü tanıyan spesifik antikorların saptanması, bireylerde ve topluluklarda enfeksiyon ve bağışıklığın değerlendirilmesi için bir araç olarak giderek daha önemli hale gelecektir. Bu amaçla, SARS-CoV-2 genomunu tespit etmeye yönelik nükleik asit amplifikasyon testleri ve serolojik testler kullanılmaktadır.

Within the scope of the "National Ruam Eradication Project" carried out between 2000-2001, Glanders was eradicated in our country. Unfortunately, 81 horses were culled in Türkiye in December 2019, following the detection of Epidemic in horses in Büyükada. In 2019, Glanders were reported in horses in Uşak and Bolu. No human cases have been reported. Türkiye is at risk for Glanders because of its geography. Therefore, reliable and fastly detection of Burkholderia mallei by PCR in a short time will prevent the distribution of unwanted infections that may occur in the future throughout the country. In this study; Burkholderia mallei strains isolated from horses were verified and optimized by PCR. The use of PCR for the detection of Burkholderia mallei was performed for the first time in our country. It has been concluded that the PCR as a diagnostic method with high reliability and sensitivity safely used together with diagnosis of Glanders.

Objectives: The incidence of MRSA still remains an important public health problem. This study aimed to perform in silico analysis of MLST, SCCmec, spa type, evolutionary similarity, and whole-genome sequencing (WGS) based antimicrobial susceptibility testing by using genomic data of MRSA strains isolated from human infections in different countries. Methods: WGS data of 30 MRSA strains were obtained as etiological agents were download from NCBI. Phylogeny analysis with large data was performed via CSI Phylogeny online software. SCCmec, MLST, and spa typing were performed using the software at the Center for Genomic Epidemiology. ResFinder 4.0 was used to perform WGS based antimicrobial susceptibility testing. Results: After in silico analysis of 30 MRSA strains, 14 different spa types, 11 different sequence types, and 9 different SCCmec types were detected. T037, ST239, and SCCmec_type_III(3A) were the most detected spa, MLST, and SCCmec types. WGS based antimicrobial susceptibility testing results were analyzed, 28, 27, and 26 out of 30 MRSA strains carrying aminoglycoside tetracycline and fluoroquinolone resistance genes respectively. Conclusion: Our in silico analysis results, we found that similar typing profiles could be observed in the strains in different geographical locations, and certain types of spa, MLST, and SCCmec can coexist.

Salmonella Typhimurium, dünyada en sık izole edilen Salmonella serotiplerinden birisidir. Tüm genom analizi gibi yeni moleküler yöntemlerin gelişmesi ile, antibiyotik direnci ve farklı genlerin karşılaştırmalı analizleri daha kolay yapılabilmektedir. Çalışmamızda farklı S. Typhimurium kökenlerine ait genomik verilerin, taşıdıkları patojenite adaları, MLST, seqserotipleri ve direnç genleri açısından karşılaştırmalı in silico analizini gerçekleştirmeyi amaçladık. Çalışmamıza genomik verileri NCBI’da yer alan 15 farklı S. Typhimurium kökenini dahil edildi. Kökenlerin birbirleri ile evrimsel olarak yakınlıkları CSI filogeni ile antimikrobiyal direnç belirteçlerinin varlığı ise ResFinder yazılımı ile gerçekleştirildi. Salmonella kökenlerine ait patojenite adaları, MLST ve serotip tespiti için sırasıyla SPIFinder, MLST ve SeqSero yazılımları kullanıldı. Çalışmamıza dahil edilen 15 S. Typhimurium kökeninin birbirlerine %97 ve üstünde genomik benzerlik gösterdikleri tespit edildi. MLST analizlerine göre 9 köken (%60) ST19 olarak bulundu. Tüm kökenlerin SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-12, SPI-13, SPI-14 patojenite adalarını taşıdıkları gözlenirken, SGI1 patojenite adası sadece 3 kökende tespit edildi. Aminoglikozid direnci ile ilişkili aac(6')-Iaa geni tüm kökenlerde saptandı. Yeni nesil dizileme tekniklerinin gelişimi, S. Typhimurium gibi önemli enfeksiyon etkenlerine ait yeni moleküler epidemiyolojik veriler elde edilmesini sağlamaktadır. Bu verilerin karşılaştırmalı in silico analizleri ile enfeksiyonlarda kullandıkları virülans genlerinin analizleri ve moleküler patogenez mekanizmalarının aydınlatılması sağlanabilecektir., Salmonella Typhimurium is one of the most frequently isolated Salmonella serotypes in the world. With the path of the new strategy of analysis like the whole genome, comparative analyzes of weaknesses and different genes can be done more easily. In our study, S. Typhimurium has different origins, it is aimed for different purposes to analyze genomic data, calculus pathogens islands, belonging MLST, seqs types, and silico genes. Our study included 15 different S. Typhimurium strains whose local data were obtained in NCBI. Evolutionary affinities of origins with C. Pathogenicity islands of Salmonella origin, SPIFinder, MLST, and SeqSero software for MLST. It was determined that Typhimurium origins showed 97% and more genomic similarity. According to MLST analysis, origin 9 (60%) was found to be ST19. While all were observed to carry pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-12, SPI-13, SPI-14, only 3 were detected in the SGI1 pathogenicity island. The aminoglycoside-related aac(6')-Iaa gene was detected in all strains. It would seem that the course of sequencing techniques such as the next generation is to obtain a new epidemiological route for owners of S. Typhimurium. With these data comparative analyzes, virulence genes that can be applied and the analyzes that can be corrected can be elucidated.

Due to their ability to resist endpoint antimicrobials such as carbapenem, it is very important to detect and monitor multi-drug resistant Gram negative strains with plasmids containing genes such as blaNDM-5 by new molecular methods. This study aimed to perform in silico analysis of resistance gene identifiers on human and animal-derived blaNDM-5 plasmids found in open databases, which were analyzed by new whole genome sequencing techniques and to compare these resistance genes. The plasmid genomic sequences of 4 human and 2 animal E. coli strains containing blaNDM-5 genes included in our study were analyzed in Silico using the Resistance Gene Identifier (RGI) option of the comprehensive antibiotic resistance gene database using default values. Human and animal strains included in our study were found to have different antimicrobial resistance genes in addition to blaNDM-5. All plasmids were found to have at least 8 perfect antimicrobial resistance gene sequences matches. When the resistance gene identifiers in all plasmids were examined, 35 resistance gene identifiers were found. Besides blaNDM-5, mphA, qacEdelta1 and sul1 were found in all plasmids. As a conclusion, it was determined by our study results that, regardless of the source, there may be different antimicrobial resistance gene identifiers besides the blaNDM-5 resistance gene in plasmids. We are of the opinion that routine molecular surveillance studies should be carried out considering the one health approach of Gram-negative pathogens such as E. coli, which can contain plasmids that cause multi-drug resistance and can be isolated from all sources., Karbapenem gibi son nokta antimikrobiyallere direnç yetenekleri dolayısıyla, blaNDM-5 gibi genler ihtiva eden plazmidlere sahip çoklu ilaç direnci gösteren Gram negatif kökenlerin yeni moleküler yöntemlerle tespiti önemlidir. Biz de çalışmamızda bu yeni tekniklerle tüm genom sekans analizi yapılan ve açık veritabanlarında bulunan insan ve hayvan kaynaklı blaNDM-5 plazmidleri üstünde bulunan direnç gen tanımlayıcılarının in silico analizini yapmayı ve bu direnç genlerini karşılaştırmayı amaçladık. Çalışmamıza dahil edilen 4 insan ve 2 hayvan kaynaklı, blaNDM-5 geni içeren E.coli kökenlerine ait plazmid genomik dizileri, varsayılan değerler kullanılarak kapsamlı antibiyotik direnç gen veritabanının, direnç geni tanımlayıcı (RGI) seçeneği kullanılarak bilgisayar ortamında analiz edildi. Çalışmamıza dahil edilen insan ve hayvan kaynaklı kökenlerde blaNDM-5 yanında farklı antimikrobiyal direnç genlerinin de olduğu tespit edildi. Tüm plazmidlerin en az 8 mükemmel antimikrobiyal direnç gen dizisi eşleşmesi gösterdiği saptandı. Tüm plazmidlerde bulunan direnç gen tanımlayıcıları incelendiğinde 35 direnç gen tanımlayıcısı saptandı. blaNDM-5 yanında, mphA, qacEdelta1 ve sul1’in bütün plazmidlerde olduğu tespit edildi. Sonuç olarak, kaynak fark etmeksizin, plazmidlerde, blaNDM-5 direnç geni yanında farklı antimikrobiyallere direnç gen tanımlayıcılarının da olabildiği çalışma sonucunda tespit edilmiştir. Çoklu ilaç direncine neden olan plazmidleri ihtiva edebilen ve tüm kaynaklardan izole edilebilen E. coli gibi Gram negatif patojenler üzerinde tek sağlık yaklaşımı düşünülerek rutin moleküler surveyans çalışmalarının yapılması gerektiği kanaatindeyiz.

Staphylococcus aureus (S. aureus) kommensal bir bakteri olarak hem insanla beraber yaşamakta, hem de fırsatçı olarak uygun koşulları bulduğunda salgıladığı bazı enzim ve toksinler nedeniyle çok ciddi ölümcül infeksiyonlara neden olabilmektedir. Bu nedenle günümüzde S. aureus hastane ve toplum kaynaklı enfeksiyonlara neden olan en önemli patojenlerden biridir. Metisilin dirençli S. aureus (MRSA) kökenleri birçok antibiyotiğe karşı dirençlidirler ve tedavi süresince kullanılan günümüz antibiyotiklerine karşı çok hızlı bir şeklide direnç geliştirme kabiliyetine sahiptirler. Bundan dolayıdır ki MRSA enfeksiyonları normal S. aureus enfeksiyonlarına oranla daha şiddetli ve inatçı enfeksiyonlara neden olmaktadırlar. S. aureus patojenitesinde MRSA kadar önemli olan diğer bir etken, özellikle ölümcül nekrotizan pnömonilerden ve toplum kökenli yumuşak deri doku enfeksiyonlarından izole edilen S.aureus suşları tarafından salgılanan Panton-Valentine lökosidin (PVL) toksinidir. PVL özellikle toplum kökenli S.aureus suşlarında gözlenmektedir. Ancak yapılan çalışmalar PVL pozitif kökenlilerin hastane ortamlarına hızla yayılmaya başladığını göstermiştir. Bu çalışmada stafilokok enfeksiyonları için en yüksek risk grubunda yer alan hastane personelinden alınan nazal swap örneklerinde nazal S.aureus taşıyıcılığı (NSAT) oranının araştırılması ile rt-PCR ile Metisiline Duyarlı S. aureus, Metisiline Dirençli Koagülaz Negatif Stafilokok ve Panton-Valentine lökosidin virülans genlerin taramasının yapılması amaçlanmıştır. Devlet hastanesinde çalışan personelden alınan nazal swap örneklerinden klasik NSAT taraması yapılmış 98 sağlık personelinin 17’sinde (%17,35) nazal S.aureus taşıyıcılığı (4’ü MSSA, 13’ü MRSA ) saptanırken ,48’inde MR-KNS tespit edilmiştir. Ayrıca alınan nazal swablardan direk olarak yapılan RT-PCR örneklerinde kültür metodu ile birebir uyumlu olarak 61 mecA(+), 17 nuc(+), 1 pvl(+) genleri tespit edilmiştir., Staphylococcus aureus (S.aureus) can cause serious fatal infections due to several enzymes and toxins that it secretes when it encounters favorable conditions.For this reason, S.aureus is one of the most important pathogens currently causing hospital and community infections.Methicillin-resistant S.aureus(MRSA) strains are resistant to many antibiotics and have the ability to rapidly develop resistance to currently used antibiotics during treatment. MRSA origins cause serious infections with high mortality and also increase the rate of hospital infections that require difficult and longer hospitalization and the use of greater quantities of antibiotics in risk-prone units in hospitals. Another factor that is as important as MRSA in S.aureus pathogenicity is the Panton-Valentine leukocidin(PVL) toxin secreted by S.aureus strains, most commonly isolated from deadly necrotizing pneumonias and community-acquired soft skin tissue infections.PVL is especially observed in community-acquired S.aureus strains. However, studies showed that PVL-positive origins began to spread rapidly to hospital environments. In this study, methicillin-sensitive S.aureus, methicillin-resistant coagulase-negative Staphylococcus (MR-CNS), and Panton-Valentine leukocidine genes were identified by real-time polymerase chain reaction (RT-PCR) by investigating the rate of nasal S.aureus carriers(NSAT) in nasal swab samples taken from hospital staff belonging to the highest risk group for staphylococcal infections, at which screening is aimed. Classical NSAT scans were performed on nasal swap samples taken from 98 healthcare professionals in Hospital. Nasal S.aureus (4 MSSA,13 MRSA) was detected in 17 samples(17.35%). During detection, 48 MR-CNS were detected. In addition, 61 mecA (+),17 nuc (+),1 pvl (+) genes were detected in RT-PCR samples directly from the nasal swabs taken in accordance with the culture method.

Homelessness has not previously been identified as a risk factor for respiratory syncytial virus (RSV) infection. We conducted an observational study at an urban safety-net hospital in Washington, USA, during 2012–2017. Hospitalized adults with RSV were more likely to be homeless, and several clinical outcome measures were worse with RSV than with influenza.

The active roles of microribonucleic acids (miRNAs) in gene regulation have made miRNAs a key point for the scientific world in the study of physiological processes. Although saliva includes the largest number of miRNAs, there is no miRNA study in saliva on horses has been found. Our study is the first study on miRNAs isolation from saliva in horses. In the present study, saliva was studied in Arabian racehorses to better understand the molecular mechanisms of expression levels that are effective in lipid metabolism of miRNAs and their target genes during the race. Identification of lipid metabolism of miRNAs and their target genes is an opportunity to provide information about biomarkers in Arabian racehorses on energy supply for race performance. Arabian racehorses have low glycogen content and high triglyceride storage capability, thanks to the high amount of oxidative type I fiber in their muscle tissue. Therefore, Arabian racehorses can provide higher levels of energy using more fat. The aim of this study is to determine the prerace and postrace expression levels of eight miRNAs in saliva that are known to affect lipid metabolism in Arabian racehorses. The expression level of eca-miR-33a was found to be statistically significant (P < .05). Target genes of eca-miR-33a have been copredicted as ABCA1, CROT, ABHD2, and SATB2, with three validated databases and other analysis tools. In conclusion, these findings revealed that both eca-miR-33a and its target genes could be potential core genes that play important roles in lipid metabolism in Arabian racehorses to provide energy during the race.

The fission yeast Schizosaccharomyces pombe has been successfully used as a model to gain fundamental knowledge in understanding how eukaryotic cells acquire copper during vegetative growth. These studies have revealed the existence of a heteromeric Ctr4–Ctr5 plasma membrane complex that mediates uptake of copper within the cells. Furthermore, additional studies have led to the identification of one of the first vacuolar copper transporters, Ctr6, as well as the copper-responsive Cuf1 transcription factor. Recent investigations have extended the use of S. pombe to elucidate new roles for copper metabolism in meiotic differentiation. For example, these studies have led to the discovery of Mfc1, which turned out to be the first example of a meiosis-specific copper transporter. Whereas copper-dependent transcriptional regulation of the Ctr family members is under the control of Cuf1 during mitosis or meiosis, meiosis-specific copper transporter Mfc1 is regulated by the recently discovered transactivator Mca1. It is foreseeable that identification of novel meiotic copper-related proteins will serve as stepping stones to unravel fundamental aspects of copper homoeostasis.

The Rhodobacter capsulatus cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes ( ccoGHIS ). It also relies on the c -type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb 3 -Cox subunits (CcoO and CcoP) are c -type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb 3 -Cox unless the growth medium is supplemented with copper. In this work, we isolated “Cu-unresponsive” derivatives of a ccoA deletion strain that exhibited no cbb 3 -Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or ccmF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb 3 -Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c 1 and c 2 than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c -type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of R. capsulatus cbb 3 -Cox proceeds independently of that of the c -type cytochromes during the biogenesis of this enzyme.

International Journal of Paediatric Dentistry 2011; 21: 29–34 Objective. The aim of this in vitro study was to evaluate the effects of using only phosphoric acid or a self-etch bonding agent under clear and opaque fissure sealants on laser fluorescence (LF) readings and the reproducibility of the laser device. Methods. Eighty extracted permanent molars, ranged from sound to carious, were randomly divided into four groups: phosphoric acid + opaque sealant (group I), Clearfil S3 Bond (Kuraray, Kurashiki, Japan) + opaque sealant (group II), phosphoric acid + clear sealant (group III), and Clearfil S3 Bond + clear sealant (group IV). The teeth were measured using an LFpen device, before and after sealing. Data were analysed using the Spearman’s correlation, Wilcoxon signed rank, and Mann–Whitney U-test. Results. Except group IV, there was a statistically significant decrease in fluorescence after the application of sealants (P 0.05). Conclusions. There was a statistically significant decrease in fluorescence for both clear and opaque sealant groups. However, clear sealant with Clearfil S3 Bond does not influence the LFpen readings.

Previously, an unexplained subcellular localization was reported for a functional fluorescent protein fusion to the response regulator OmpR in Escherichia coli . The pronounced regions of increased fluorescence, or foci, are dependent on OmpR phosphorylation and do not occupy fixed, easily identifiable positions, such as the poles or mid-cell. Here we show that the foci are due to OmpR-YFP (yellow fluorescent protein) fusion binding to specific sites in the chromosome. To identify positions of foci and quantify their fluorescence intensity, we used a simple system to tag virtually any chromosomal location with arrays of lacO or tetO . The brightest foci colocalize with the OmpR-regulated gene ompF , which is strongly expressed under our growth conditions. When we increased OmpR-YFP phosphorylation by stimulating the EnvZ/OmpR system with procaine, we observed a small increase in OmpR-YFP fluorescence at ompF and a significant increase at the OmpR-regulated gene ompC . This supports a model of hierarchical binding of OmpR to the ompF and ompC promoters. Our results explain the inhomogeneous distribution of OmpR-YFP fluorescence in cells and further demonstrate that for a transcription factor expressed at wild-type levels, binding to native sites in the chromosome can be imaged and quantified by fluorescence microscopy.

Cytochromes (cyts) are ubiquitous heme containing proteins that are key components of energy transduction pathways. They participate in a wide variety of electron transfer reactions, which are essential for cellular processes responsible for chemical energy (ATP) production. The cbb3-type cyt c oxidase (cbb3-Cox) provides an excellent model to study biogenesis of membrane-integral, oligomeric cyt c complexes. Its subunits contain three hemes c, two hemes b and a copper (CuB) atom as cofactors that use distinct insertion processes. In cyts c, heme b is covalently ligated (referred to as heme c) via a complex maturation process that involves in some species up to nine components (Ccm-System I). In addition to the cyts c, many cyt c containing complexes carry other cofactors, and insertion of these cofactors requires additional biogenesis components besides the Ccm-system I. In the case of cbb3-Cox, the mechanisms underlying incorporation of hemes b into the catalytic subunit are not well understood. However, remarkable progress was achieved recently on how the single CuB atom at the catalytic heart of this heme-copper oxidase is acquired. Finally, insertion of the cofactors must be temporally and spatially coordinated with the assembly of the subunits in order to yield a functional cbb3-Cox enzyme. In this chapter, we discuss the biogenesis of cbb3-Cox from the insertion of its catalytic heme-copper (CuB) center and maturation of its c-type cyts to the assembly of its mature subunits, mainly focusing on studies carried out with the anoxygenic phototrophic bacterium Rhodobacter capsulatus.

MikroRNA’lar (miRNA)18-25 nükleotit (nt) uzunluğunda genom üzerinde protein kodlayan intron veya ekzon bölgelerindeki RNA genlerinden transkripsiyonu sağlanan, fakat proteine translasyonu gerçekleşmeyen, fonksiyonel RNA molekülleridir. Tüm canlılarda çeşitli fiziksel ve patolojik kondisyonlarda post–transkripsiyonel gen düzenleyici olarak miRNA’ların rolü evcil hayvanlar da dahil birçok organizmanın incelenmesiyle aydınlanmaktadır. miRNA’ların gen ekspresyonunda, fenotipik değişkenliğin şekillenmesinde ve hastalık gelişminde regülatör olarak önemli rollerinin olduğu tespit edilmiştir. Yapılan çalışmaların ışığı altında miRNA’ların önemi son zamanlarda Veteriner Hekimlikte de fark edilmiştir. Taşıma ve eğlence için kullanılan, aynı zamanda ekonomik değere sahip evcil at (Equus caballus), insan uygarlığının çok önemli bir parçası olmuştur. Tıbbi açıdan at ile insan arasında alerji ve osteoartrit gibi 90’dan fazla kalıtsal hastalık ortaktır. Biyomekanik ve egzersiz fizyolojisi çalışmalarında en iyi model organizma attır. İnsanlık tarihi için at bu kadar önemli bir organizma olmasına rağmen, atlarda miRNA’lar ve miRNA’ların klinik kondüsyonlar üzerine etkisi hakkındaki çalışmalar oldukça azdır. Bu derleme miRNA’lar ve atlarda miRNA’lar ile ilgili yapılan çalışmalar hakkında bilgi vermek amacıyla hazırlanmıştır.

Copper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb3-type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb3-Cox production in bacteria like R. capsulatus., IMPORTANCE Copper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochrome c oxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochrome c oxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochrome c oxidase in the cell.

The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb3-type Cox (cbb3-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb3-Cox defective mutant that requires Cu2+ supplementation to produce an active cbb3-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb3-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb3-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition., IMPORTANCE Biogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochrome c oxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototroph Rhodobacter capsulatus as a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content and cbb3-type cytochrome c oxidase production in bacteria.

The aim of this in vitro study was to evaluate the effects of using only phosphoric acid or a self-etch bonding agent under clear and opaque fissure sealants on laser fluorescence (LF) readings and the reproducibility of the laser device.Eighty extracted permanent molars, ranged from sound to carious, were randomly divided into four groups: phosphoric acid + opaque sealant (group I), Clearfil S3 Bond (Kuraray, Kurashiki, Japan) + opaque sealant (group II), phosphoric acid + clear sealant (group III), and Clearfil S3 Bond + clear sealant (group IV). The teeth were measured using an LFpen device, before and after sealing. Data were analysed using the Spearman's correlation, Wilcoxon signed rank, and Mann-Whitney U-test.Except group IV, there was a statistically significant decrease in fluorescence after the application of sealants (P0.05). The decrease of LFpen readings in the opaque sealant groups was more significant than the clear sealant groups (P0.05). But for both sealants, the difference between phosphoric acid and Clearfil S3 Bond groups was nonsignificant (P0.05).There was a statistically significant decrease in fluorescence for both clear and opaque sealant groups. However, clear sealant with Clearfil S3 Bond does not influence the LFpen readings.

During cytochrome c maturation (Ccm), the DsbA-dependent thio-oxidative protein-folding pathway is thought to introduce a disulphide bond into the haem-binding motif of apocytochromes c. This disulphide bond is believed to be reduced through a thio-reductive pathway involving the Ccm components CcdA (DsbD), CcmG and CcmH. Here, we show in Rhodobacter capsulatus that in the absence of DsbA cytochrome c levels were decreased and CcdA or CcmG or the putative glutathione transporter CydDC was not needed for Ccm. This decrease was not due to overproduction of the periplasmic protease DegP as a secondary effect of DsbA absence. In contrast, CcmH was absolutely necessary regardless of DsbA, indicating that compensatory thio-redox interactions excluded it. Remarkably, the double (DsbA-CcmG) and triple (DsbA-CcmG-CcdA) mutants produced cytochromes c at lower levels than the DsbA-null mutants, unless they contained a CcmG derivative (CcmG*) lacking its thio-reductive activity. Purified CcmG* can bind apocytochrome c in vitro, revealing for the first time a thiol-independent, direct interaction between apocytochrome c and CcmG. Furthermore, elimination of the thio-redox components does not abolish cytochrome c production, restricting the number of Ccm components essential for haem-apocyt c ligation per se during Ccm.

The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb(3)-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b(3)-Cu(B) center, have to be coordinated precisely both temporally and spatially to yield a functional cbb(3)-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb(3)-Cox, and provide a highly tentative model for cbb(3)-Cox assembly and formation of its heme b(3)-Cu(B) binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.