Effect of orthodontic forces on levels of enzymes in gingival crevicular fluid (GCF): A systematic review.

OBJECTIVE: Orthodontic force application releases multiple enzymes in gingival crevicular fluid (GCF) for activation, resorption, reversal, deposition of osseous elements and extracellular matrix degradation. The current systematic review critically evaluated all existing evidence on enzymes in orthodontic tooth movement. METHODS: Literature was searched with predetermined search strategy on electronic databases (PubMed, Scopus, Embase), along with hand search. RESULTS: Initial search identified 652 studies, shortlisted to 52 studies based on PRISMA. Quality assessment further led to final inclusion of 48 studies (13 moderately and 35 highly sensitive studies). Primary outcomes are significant upregulation in GCF levels of enzymes-aspartate aminotransferase (AST), alkaline phosphatase (ALP), matrix metalloproteinases (MMPs), lactate dehydrogenase (LDH), ß-glucuronidase (ßG), tartrate resistant acid phosphatase (TRAP), acid phosphatase (ACP) and down regulation in cathepsin B (Cb). Site specificity is shown by ALP, TRAP, AST, LDH, MMP9 with levels at compression site increasing earlier and in higher quantities compared with tension site. ALP levels are higher at tension site only in retention. A positive correlation of LDH, ALP and AST is also observed with increasing orthodontic force magnitude. CONCLUSIONS: A strong evidence of variation in enzymes (ALP, AST, ACP TRAP, LDH, MMPs, Cb) in GCF is found in association with different magnitude, stages and sites of orthodontic force application.

Effect of orthodontic forces on levels of enzymes in gingival crevicular fluid (GCF): A systematic review

Abstract Objective: Orthodontic force application releases multiple enzymes in gingival crevicular fluid (GCF) for activation, resorption, reversal, deposition of osseous elements and extracellular matrix degradation. The current systematic review critically evaluated all existing evidence on enzymes in orthodontic tooth movement. Methods: Literature was searched with predetermined search strategy on electronic databases (PubMed, Scopus, Embase), along with hand search. Results: Initial search identified 652 studies, shortlisted to 52 studies based on PRISMA. Quality assessment further led to final inclusion of 48 studies (13 moderately and 35 highly sensitive studies). Primary outcomes are significant upregulation in GCF levels of enzymes-aspartate aminotransferase (AST), alkaline phosphatase (ALP), matrix metalloproteinases (MMPs), lactate dehydrogenase (LDH), β-glucuronidase (βG), tartrate resistant acid phosphatase (TRAP), acid phosphatase (ACP) and down regulation in cathepsin B (Cb). Site specificity is shown by ALP, TRAP, AST, LDH, MMP9 with levels at compression site increasing earlier and in higher quantities compared with tension site. ALP levels are higher at tension site only in retention. A positive correlation of LDH, ALP and AST is also observed with increasing orthodontic force magnitude. Conclusions: A strong evidence of variation in enzymes (ALP, AST, ACP TRAP, LDH, MMPs, Cb) in GCF is found in association with different magnitude, stages and sites of orthodontic force application.

Resumo Objetivo: a aplicação da força ortodôntica libera múltiplas enzimas no fluído crevicular gengival (FCG), desencadeando a ativação, reabsorção, reversão, deposição de elementos ósseos e degradação da matriz extracelular. A presente revisão sistemática avaliou criticamente toda a evidência disponível sobre os níveis de enzimas durante a movimentação ortodôntica. Métodos: utilizando-se estratégias predeterminadas, foram realizadas buscas em bases de dados eletrônicas (PubMed, Scopus, Embase), sendo também feitas buscas manuais. Resultados: a busca inicial identificou 652 estudos e, com base nas diretrizes do PRISMA, foram selecionados 52 estudos. A avaliação qualitativa resultou na inclusão final de 48 estudos (13 estudos com moderada sensibilidade e 35 com alto nível de sensibilidade). Os desfechos primários foram o aumento significativo dos níveis no FCG das enzimas aspartato aminotransferase (AST), fosfatase alcalina (FA), metaloproteinases de matriz (MMPs), lactato desidrogenase (LDH), β-glucuronidase (βG), fosfatase ácido-resistente ao tartarato (TRAP), fosfatase ácida (FAC) e baixa regulação de catepsina B (Cb). Especificidade quanto ao local foi mostrada para FA, TRAP, AST, LDH e MMP9 com os níveis no lado de compressão aumentando mais rápido e em maiores quantidades, quando comparado ao lado de tensão. Os níveis de FA foram maiores no lado de tensão somente no período de contenção. Uma correlação positiva de LDH, FA e AST também foi observada à medida que a magnitude de força ortodôntica aumentou. Conclusões: há fortes evidências indicando que as variações nas enzimas (FA, AST, FAC, TRAP, LDH, MMPs, Cb) presentes no FCG estão associadas a diferentes magnitudes, estágios e locais de aplicação da força ortodôntica.

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported.

Conformational stability of OXA-51 ß-lactamase explains its role in carbapenem resistance of Acinetobacter baumannii.

Acinetobacter baumannii, an important nosocomial pathogen, is increasingly becoming resistant to antibiotics including recent ß-lactam like imipenem. Production of different types of ß-lactamases is one of the major resistance mechanisms which bacteria adapt. We recently reported the presence of a ß-lactamase, OXA-51, in clinical strains of A. baumannii in ICUs of our hospital. This study is an attempt to understand the structure-function relationship of purified OXA-51 in carbapenem resistance in A. baumannii. The OXA-51 was cloned, expressed in E. coli Bl-21(DE3) and further purified. The in vitro enzyme activity of purified OXA-51 was confirmed by two independent techniques; in-gel assay and spectrophotometric method using nitrocefin. Further in vivo effect of OXA-51 was followed by transmission electron microscopy of bacterium. Biophysical and biochemical investigations of OXA-51 were done using LC-MS/MS, UV-Vis absorption, fluorescence, circular dichroic spectroscopy and isothermal calorimetry. Native OXA-51 was characterized as 30.6 kDa, pI 8.43 with no disulphide bonds and comprising of 30% α-helix, 27% ß-sheet. Secondary structure of OXA-51 was significantly unchanged in broad pH (4-10) and temperature (30-60 °C) range with only local alterations at tertiary structural level. Interestingly, enzymatic activity up to 75% was retained under above conditions. Hydrolysis of imipenem by OXA-51 (k(m),1 µM) was found to be thermodynamically favourable. In the presence of imipenem, morphology of sensitive strain of A. baumannii was drastically changed, while OXA-51-transformed sensitive strain retained the stable coccobacillus shape, which demonstrates that imipenem is able to kill sensitive strain but is unable to do so in OXA-51-transformed strain. Hence the production of pH- and temperature-stable OXA-51 appears to be a major determinant in the resistance mechanisms adopted by A. baumannii in order to evade even the latest ß-lactams, imipenem. It can be concluded from the study that OXA-51 plays a vital role in the survival of the pathogen under stress conditions and thus poses a major threat.

Quantitative profiling and identification of plasma proteins of spinocerebellar ataxia type 2 patients.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is an autosomal-dominant hereditary ataxia characterized by progressive gait and limb ataxia, dysarthria, slow saccades, neuropathy and dementia. The expansion of trinucleotide CAG repeats in the coding region of the ATXN-2 gene leads to expanded polyglutamine stretch in the mutated protein which causes neuronal death. OBJECTIVE: In this study, we investigated the blood plasma of SCA2 patients to find protein biomarkers. METHODS: Thirty-two ataxia patients clinically suspected for SCA2 were evaluated by the International Co-operative Ataxia Rating Scale followed by genetic analysis using PCR. Plasma proteomics of SCA2 patients and age- and gender-matched healthy controls was done using 2D-difference in-gel electrophoresis, LC-MS/MS and Western blot. RESULTS: Genetic analysis confirmed 10 of 32 suspected SCA2 patients. Proteomic data revealed nine differentially expressed proteins in SCA2. These proteins find good association with oxidative stress, calcium-dependent apoptosis, neuropathy, and cognitive impairment in SCA2 patients. Interestingly, the elevated levels of the voltage-dependent calcium channel γ-3 subunit showed a direct correlation with calcium-generated apoptosis of Purkinje cells. The cognitive deficit, a common symptom in SCA2 patients, seems to correlate with decreased levels of transthyretin and retinol-binding protein-4. CONCLUSIONS: Some of these identified proteins in SCA2 can be useful for therapeutic, diagnostic and prognostic purposes.

Structural studies on New Delhi Metallo-ß-lactamase (NDM-2) suggest old ß-lactam, penicillin to be better antibiotic for NDM-2-harbouring Acinetobacter baumanni.

Acinetobacter baumannii, a Gram-negative pathogen causes nosocomial infections including pneumonia, urinary tract and respiratory infections. Carbapenem group of ß-lactam antibiotics are routinely used to treat A. baumannii including multidrug-resistant clinical strains. The emergence of New Delhi Metallo-ß-lactamase (NDM-2), a new type of ß-lactamase and one of the major resistant determinants in A. baumannii, opened up challenges in the treatment of resistant strains. Thus, understanding the structure-function relationship of NDM-2 with different analogues of ß-lactams becomes crucial. We carried out in silico studies on the interaction of various ß-lactams with NDM-2 and with OXA-24, a carbapenem hydrolyzing non-NDM type ß-lactamase. The binding affinity of the ß-lactams to NDM-2 was found to be in the order: ceftazidime ≈ imipenem ≈ doripenem > oxacillin > aztreonam > penicillin; however, the order of their affinity to OXA-24 was quite different: ceftazidime > aztreonam > penicillin > oxacillin > doripenem > imipenem. Further, NDM-2 in comparison to OXA-24 showed stronger interaction (less X-score) with most of the ß-lactams except penicillin. This suggests higher lethality posed by clinical strains expressing NDM-2 than those without NDM-2. Weak interaction between NDM-2 and penicillin clearly points out that penicillin is perhaps better option in treating A. baumannii harbouring NDM-2. Present findings provide new insights in drug resistance at the molecular level of NDM-2 and can help in designing structure-based drugs.

Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain.

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A.baumannii even to the latest generation of ß-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A.baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A.baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A.baumannii are: i) ß-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A.baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A.baumannii to carbapenem.

Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii isolated from India.

Acinetobacter baumannii, a Gram negative bacterium causes nosocomial infections including bacteremia, secondary meningitis and urinary tract infections. Increased resistance of A. baumannii has been global concern. Till recently, carbapenems, latest generation of ß-lactams are used for treating infections caused by A. baumannii. Emerging resistance to carbapenem class is an immediate threat to mankind. The objective of present study is to understand the growing carbapenem resistance of A. baumannii. By using iso-electric focusing followed by (in-gel) nitrocefin assay of carbapenem resistant strains of A. baumannii, we could identify three ß-lactamases with pIs in the range 5.4-9.5. Expression of the ß-lactamase with a pI ≈ 8.5, was found only in very high carbapenem resistant (MIC for imipenem 128 µg/ml) strains. On PCR analysis and sequencing of PCR product, this ß-lactamase was confirmed to be OXA-51. Identification of this protein from IEF gel was reconfirmed with the help of Liquid chromatography and Tandem mass spectrometry (LC-MS/MS). Based on the amino acid sequence, OXA-51 found to be a 30 kDa ß-lactamase containing conserved functional motifs of class D serine ß-lactamase. In the present study, we have established the emergence of OXA-51 in clinical strains of A. baumannii in India which suggests its role in carbapenem resistance.

In-silico modeling of a novel OXA-51 from ß-lactam-resistant Acinetobacter baumannii and its interaction with various antibiotics.

Acinetobacter baumannii, one of the major Gram negative bacteria, causes nosocomial infections such as pneumonia, urinary tract infection, meningitis, etc. ß-lactam-based antibiotics like penicillin are used conventionally to treat infections of A. baumannii; however, they are becoming progressively less effective as the bacterium produces diverse types of ß-lactamases to inactivate the antibiotics. We have recently identified a novel ß-lactamase, OXA-51 from clinical strains of A. baumannii from our hospital. In the present study, we generated the structure of OXA-51 using MODELLER9v7 and studied the interaction of OXA-51 with a number of ß-lactams (penicillin, oxacillin, ceftazidime, aztreonam and imipenem) using two independent programs: GLIDE and GOLD. Based on the results of different binding parameters and number of hydrogen bonds, interaction of OXA-51 was found to be maximum with ceftazidime and lowest with imipenem. Further, molecular dynamics simulation results also support this fact. The lowest binding affinity of imipenem to OXA-51 indicates clearly that it is not efficiently cleaved by OXA-51, thus explaining its high potency against resistant A. baumannii. This finding is supported by experimental results from minimum inhibitory concentration analysis and transmission electron microscopy. It can be concluded that carbapenems (imipenem) are presently effective ß-lactam antibiotics against resistant strains of A. baumannii harbouring OXA-51. The results presented here could be useful in designing more effective derivatives of carbapenem.