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1.  Initial experience with GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program 
The Australasian Medical Journal  2014;7(5):203-207.
Background
Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. Rapid and accurate diagnosis of pulmonary and extra-pulmonary tuberculosis (TB) is still a great challenge. The GeneXpert MTB/RIF assay is a novel integrated diagnostic system for the diagnosis of tuberculosis and rapid detection of Rifampin (RIF) resistance in clinical specimens. In 2012, the Arkansas Tuberculosis Control Program introduced GeneXpert MTB/RIF assay to replace the labour-intensive Mycobacterium Tuberculosis Direct (MTD) assay.
Aims
To rapidly diagnose TB within two hours and to simultaneously detect RIF resistance.
Objectives
Describe the procedure used to introduce GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.
Characterise the current gap in rapid M. tuberculosis diagnosis in Arkansas.
Assess factors that predict acid fast bacilli (AFB) smearnegative but culture-positive cases in Arkansas.
Illustrate, with two case reports, the role of GeneXpert MTB/RIF assay in reduction of time to confirmation of M. tuberculosis diagnosis in the first year of implementation.
Method
Between June 2012 and June 2013, all AFB sputum smearpositive cases and any others, on request by the physician, had GeneXpert MTB/RIF assay performed as well as traditional M. tuberculosis culture and susceptibilities using Mycobacteria Growth Indicator Tube (MGIT) 960 and Löwenstein-Jensen (LJ) slants. Surveillance data for January 2009–June 2013 was analysed to characterise sputum smear-negative but culture-positive cases.
Results
Seventy-one TB cases were reported from June 2012– June 2013. GeneXpert MTB/RIF assay identified all culture-positive cases as well as three cases that were negative on culture. Also, this rapid assay identified all six smear-negative but M. tuberculosis culture-positive cases; two of these cases are described as case reports.
Conclusion
GeneXpert MTB/RIF assay has made rapid TB diagnosis possible, with tremendous potential in determining isolation of TB suspects on one hand, and quickly ruling out TB whenever suspected.
doi:10.4066/AMJ.2014.1905
PMCID: PMC4052441  PMID: 24944716
Tuberculosis; GeneXpert MTB/RIF assay; AFB
2.  Role of mitochondrial-derived oxidants in renal tubular cell cold storage injury 
Free radical biology & medicine  2010;49(8):1273-1282.
Cold storage (CS) is regarded as a necessary procedure during donation of a deceased donor kidney that helps to optimize organ viability. Increased oxidant generation during both CS as well as during the reperfusion (or rewarming/CS.RW) phase have been suggested to be a major contributor to renal injury; although the source and/or biochemical pathways involved with oxidant production remain unclear. The purpose of this study was to determine if renal tubular mitochondrial superoxide is capable of inducing oxidant production and mitochondrial damage in response to a CS.RW insult. To test the role of mitochondrial superoxide in CS.RW injury, we used rat renal proximal tubular (NRK) cells overexpressing manganese superoxide dismutase (MnSOD), the major mitochondrial antioxidant. Oxidant production, mitochondrial membrane potential, respiratory complex function, and cell death were all altered following exposure of NRK cells to CS.RW. MnSOD overexpression or inhibition of nitric oxide synthase (NOS) provided significant protection against oxidant generation, respiratory complex inactivation, and cell death. These findings implicate mitochondrial superoxide, nitric oxide, and their reaction product, peroxynitrite, as key signaling molecules involved in CS.RW injury of renal tubular cells, and suggest that therapeutic inhibition of these pathways may protect the donor kidney.
doi:10.1016/j.freeradbiomed.2010.07.012
PMCID: PMC3688469  PMID: 20659553
Cold preservation; cold storage; superoxide; nitric oxide; peroxynitrite; mitochondria; respiratory complexes
3.  Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse 
Free radical biology & medicine  2011;51(2):406-416.
Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.
doi:10.1016/j.freeradbiomed.2011.04.024
PMCID: PMC3118857  PMID: 21571061
Cre-Lox technology; Kidney; MnSOD; Cre recombinase; Superoxide; Nitrotyrosine
4.  Alteration of renal respiratory Complex-III during experimental type-1 diabetes 
Background
Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).
Methods
Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.
Results
Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.
Conclusion
In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.
doi:10.1186/1472-6823-9-2
PMCID: PMC2636815  PMID: 19166612
5.  Manganese Porphyrin Reduces Renal Injury and Mitochondrial Damage during Ischemia/Reperfusion ± 
Free radical biology & medicine  2007;42(10):1571-1578.
Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant Mn(III) meso-tetrakis(N-hexylpyridinium-2-yl)porphyrin, (MnTnHex-2-PyP5+) using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP5+ in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 hr protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 μg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified ATP synthase- beta subunit as a key protein induced by MnTnHex-2-PyP5+ treatment alone, and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP5+ protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.
doi:10.1016/j.freeradbiomed.2007.02.016
PMCID: PMC1924492  PMID: 17448904
kidney; ischemia/reperfusion; metalloporphyrin; proteomics; MnSOD; mitochondria; oxidants; nitrotyrosine; blue native polyacrylamide gel electrophoresis BN-PAGE; two dimensional fluorescence differential in gel electrophoresis (2D-DIGE)

Results 1-5 (5)