A Multinational Analysis of Mutations and Heterogeneity in PZase, RpsA, and PanD Associated with Pyrazinamide Resistance in M/XDR Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne's assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8-10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance.

Phenotypic and genotypic diversity in a multinational sample of drug-resistant Mycobacterium tuberculosis isolates.

OBJECTIVE: To develop and evaluate rapid, molecular-based drug susceptibility testing (DST) for extensively drug-resistant tuberculosis (XDR-TB), we assembled a phenotypically and genotypically diverse collection of Mycobacterium tuberculosis isolates from patients evaluated for drug resistance in four high-burden countries. METHODS: M. tuberculosis isolates from India (n = 111), Moldova (n = 90), the Philippines (n = 96), and South Africa (n = 103) were selected from existing regional and national repositories to maximize phenotypic diversity for resistance to isoniazid, rifampin (RMP), moxifloxacin, ofloxacin, amikacin, kanamycin, and capreomycin. MGIT™ 960 was performed on viable isolates in one laboratory using standardized procedures and drug concentrations. Genetic diversity within drug resistance phenotypes was assessed. RESULTS: Nineteen distinct phenotypes were observed among 400 isolates with complete DST results. Diversity was greatest in the Philippines (14 phenotypes), and least in South Africa (9 phenotypes). Nearly all phenotypes included multiple genotypes. All sites provided isolates resistant to injectables but susceptible to fluoroquinolones. Many patients were taking drugs to which their disease was resistant. DISCUSSION: Diverse phenotypes for XDR-TB-defining drugs, including resistance to fluoroquinolones and/or injectable drugs in RMP-susceptible isolates, indicate that RMP susceptibility does not ensure effectiveness of a standard four-drug regimen. Rapid, low-cost DST assays for first- and second-line drugs are thus needed.

Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using the MODS assay.

OBJECTIVE: To establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay. SETTING: A multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa. DESIGN: In the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined. In the second phase, MODS second-line drug susceptibility testing (DST) as an indirect test was compared to MGIT™ DST (n = 89). In the third (n = 30) and fourth (n = 156) phases, we determined the reproducibility and concordance of MODS second-line DST directly from sputum. RESULTS: Breakpoints for MFX (0.5 µg/ml), OFX (1 µg/ml), AMK (2 µg/ml), KM (5 µg/ml) and CPM (2.5 µg/ml) were determined. In all phases, MODS results were highly concordant with MGIT DST. The few discrepancies suggest that the MODS breakpoint concentrations for some drugs may be too low. CONCLUSION: MODS second-line DST yielded comparable results to MGIT second-line DST, and is thus a promising alternative. Further studies are needed to confirm the accuracy of the drug breakpoints and the reliability of MODS second-line DST as a direct test.

The average roughness and fractal dimension of articular cartilage during drying.

Cartilage is a unique material in part because of it biphasic properties. The structure of cartilage is a porous matrix of collagen fibers, permeated with synovial fluid which creates a gliding and near frictionless motion in articulating joints. However, during in vitro testing or surgery, there exists potential for cartilage to dehydrate, or dry out. The effects of this drying can influence experimental results. It is likely that drying also changes joint performance in vivo. In in vitro testing of equine cartilage explants exposed to open air, the average roughness of cartilage changes from 1.85 ± 0.78 to 3.66 ± 1.41 µm SD in a 5-h period. Significant changes in roughness in individual samples are seen within 10 min of exposure to open air. However, the multi-scale nature of cartilage, characterized by the fractal dimension, does not significantly change during the same period. The current study attempts to quantify the magnitude of error that is introduced when cartilage is removed from its native environment.

Diabetes and gout: efficacy and safety of febuxostat and allopurinol.

AIM: Assess influences of demographics and co-morbidities of gout patients with or without diabetes on safety and efficacy of urate-lowering agents. METHODS: Post-hoc analysis of 312 diabetic and 1957 non-diabetic gout patients [baseline serum urate levels (sUA) ≥8.0 mg/dl] enrolled in a 6-month randomized controlled trial comparing urate-lowering efficacy (ULE) and safety of daily xanthine oxidase inhibitors (XOIs) febuxostat (40 mg or 80 mg) and allopurinol (200 mg or 300 mg). We compared baseline demographic, gout and co-morbid characteristics, ULE, and safety of XOI treatment in diabetic and non-diabetic gout patients. ULE was measured by the proportion of diabetic and non-diabetic patients in each treatment group achieving final visit sUA < 6.0 mg/dl. Safety was monitored throughout the trial. RESULTS: Diabetic gout patients were older, more frequently female, and had longer gout duration. Co-morbidities were more frequent among diabetic patients: cardiovascular disease; impaired renal function; hyperlipidemia; and obesity (body mass index >30 kg/m²) (p < 0.001 for all comparisons). Febuxostat 80 mg ULE exceeded that of febuxostat 40 mg or allopurinol (p < 0.050) at all levels of renal function, achieving sUA goal range in the majority of diabetic and non-diabetic patients. Diabetics and non-diabetics reported self-limiting diarrhoea and URIs as the most common adverse events. CONCLUSIONS: Despite higher co-morbidity rates in diabetic patients, febuxostat and allopurinol were safe in both groups at the doses tested. Febuxostat 80 mg achieved sUA <6.0 mg/dl more often than febuxostat 40 mg or allopurinol at commonly prescribed doses.

Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine.

Despite the use of hearing protection devices (HPDs) and engineering changes designed to improve workspaces, noise-induced hearing loss continues to be one of the most common and expensive disabilities in the US military. Many service members suffer acoustic trauma due to improper use of HPDs, sound levels exceeding the protective capacity of the HPDs, or by unexpected, injurious exposures. In these cases, there is no definitive treatment for the hearing loss. This study investigated the use of the pharmacological agents N-acetylcysteine and acetyl-L-carnitine after acoustic trauma to treat cochlear injury. N-Acetylcysteine is an antioxidant and acetyl-L-carnitine a compound that maintains mitochondrial bio-energy and integrity. N-Acetylcysteine and acetyl-L-carnitine, respectively, significantly reduced permanent threshold shifts and hair cell loss compared to saline-treated animals when given 1 and 4 h post-noise exposure. It may be possible to obtain a greater therapeutic effect using these agents in combination or at higher doses or for a longer period of time to address the secondary oxidative events occurring 7-10 days after acute noise exposure.

Targeted topical steroid therapy in sudden sensorineural hearing loss.

OBJECTIVE: To treat patients with sudden sensorineural hearing loss (SSNL) who failed oral prednisone therapy by using a round window membrane (RWM) microcatheter. This topical delivery strategy sought to improve effectiveness of steroid treatment to the inner ear by targeting drug delivery to the RWM. STUDY DESIGN: Nonrandomized prospective design. SETTING: Tertiary care facility. PATIENTS: Six patients with severe unilateral SSHL, five of whom were refractory to a course of oral steroid therapy treated within 6 weeks of SSHL and three additional patients treated more than 6 weeks after SSHL. INTERVENTION: Therapeutic use of RWM catheter. MAIN OUTCOME MEASURES: Pure-tone averages (PTAs) and word identification scores (WIS). RESULTS: Five of the six patients treated within 6 weeks of SSHL improved their WIS. Of the six, four returned to baseline hearing, one recovered hearing that could benefit by hearing amplification, and one regained moderate improvement in PTA but not WIS. CONCLUSION: Targeted topical steroid administration avoids the significant systemic side effects of oral steroids and may offer more effective dosing than simple transtympanic injection of medicine. Although these findings are preliminary, it is possible that after further study, targeted drug delivery may be a useful technique to consider in patients with severe to profound hearing loss that have failed all other management options.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function.

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor alpha (TGFalpha), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

A genetic analysis of neural progenitor differentiation.

Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.

Reduction of noise-induced hearing loss using L-NAC and salicylate in the chinchilla.

The effects of a combination of two antioxidant compounds were studied in a chinchilla model of noise-induced hearing loss. After obtaining baseline hearing thresholds using inferior colliculus evoked potentials, chinchillas were exposed for 6 h to octave band noise centered at 4 kHz (105 dB SPL). Post-noise thresholds were obtained 1 h after the noise exposure, and then animals received either saline or salicylate and N-L-acetylcysteine combination. Another group received antioxidant treatment 1 h prior to noise. Hearing was tested at 1, 2 and 3 weeks post-noise. Subsequently, the cochleae were harvested, and cytocochleograms were prepared. There was a 20-40 dB SPL threshold shift at 3 weeks for tested controls. Permanent threshold shifts (PTS) were significantly reduced (P<0.05) to approximately 10 dB for the pre-treatment group at week 3. The PTS for the post-treatment group at week 3 was similar to the pre-treatment group at 1 and 2 kHz (0-10 dB) but was intermediate between the control and pre-treatment groups at 4 and 8 kHz (23 dB). Animals pre-treated with antioxidant had a significant reduction in hair cell loss but those post-treated with antioxidant had no protection from hair cell loss. These findings demonstrate the feasibility of reduction of noise-induced hearing loss using clinically available antioxidant compounds.

In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease.

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (inactivation rate constant (k(obs)/[I]) = 1,470,000 +/- 440,000 M(-1) s(-1) for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha(1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.

Calcium, magnesium, and phosphorus: emergency department testing yield.

OBJECTIVES: To investigate how often the ED ordering of stat serum calcium (Ca+2), magnesium (Mg+2), and phosphorus (PO4(-3)) levels affected clinical treatment; to define the diagnoses of patients for whom Ca+2, Mg+2, and PO4(-3) measurements did affect clinical therapy; and to suggest guidelines for more appropriate ordering of these laboratory tests. METHODS: A retrospective chart review was performed in an academic teaching hospital. All adult ED patients who had Ca+2, Mg+2, or PO4(-3) laboratory testing during the 9-month study period were included and evaluated for potential clinical impact of an abnormal Ca+2, Mg+2, or PO4(-3) laboratory test. RESULTS: 1.477 patients had Ca+2, Mg+2, or PO4(-3) measured while in the ED during the study period. Of these, 260 patients (17.6%) had a total of 312 abnormal Ca+2, Mg+2, or PO4(-3) values as defined by results exceeding +/- 15% of normal reference values. Of these, only 5 patients (0.3%) received treatment for abnormal values in the ED, while 75 patients (5.1%) were treated once admitted to the hospital. In this study, the only diagnostic groups to whom significant treatment was administered were diabetic patients (Ca+2 and PO4(-3); alcoholic patients (Mg+2); and renal failure patients (Ca+2, Mg+2, and PO4(-3). CONCLUSION: These results suggest that stat Ca+2, Mg+2, and PO4(-3) levels seldom affect clinical treatment in the ED. The frequency of ordering these tests may be reduced by obtaining Ca+2, Mg+2, or PO4(-3) measurements only for patients known to be at risk for such abnormalities, based on their existing or suspected diagnoses. The authors suggest obtaining these tests, when indicated, on a "non-stat" basis, with the subsequent laboratory results becoming available in-hospital, where treatment is more likely to occur.

Isolation and characterization of vesicular stomatitis virus PoIR revertants: polymerase readthrough of the leader-N gene junction is linked to an ATP-dependent function.

The switch from transcription to replication of the VSV genome is coupled to assembly of nascent chains and involves an unspecified change in the P-L polymerase complex when it reaches the leader-N gene junction. PoIR VSV mutants, in contrast to wild-type virus, read through this first gene junction at high frequency without concurrent assembly, and they show altered ATP requirements for transcription in vitro. The mutation(s) responsible for the poIR phenotype segregates to the N-RNA template fraction. We report here that both poIR1 and poIR2 mutants display severe growth restriction in mouse L cells but not in BHK cells. Four of six poIR1 revertant viruses, originating from rare plaques on L cells, showed wild-type characteristics for growth, readthrough of leader-N gene junction, and ATP utilization, while two showed partial and quantitatively parallel coreversion of all properties. Sequence analysis of N and P genes of poIR mutants and revertants provided proof that a single mutation in the N protein, Arg179 to His, is responsible for the poIR phenotype. PoIR1, but not poIR2, also displayed a phenotypically silent GA-to-GG change in the N-P intergenic dinucleotide sequence Five of six revertants retained the poIR1 N protein mutation and showed no change in their P gene. We conclude that the L protein likely contains second-site suppressors of the poIR phenotype, and we propose that the switch from transcription to replication is modulated by an ATP-dependent interaction between the template-associated N protein and the L subunit of the P L polymerase complex.

Constitutive phosphorylation of the vesicular stomatitis virus P protein modulates polymerase complex formation but is not essential for transcription or replication.

As a subunit of both the P-L polymerase complex and the P-N assembly complex, the vesicular stomatitis virus (VSV) P protein plays a pivotal role in transcription and replication of the viral genome. Constitutive phosphorylation of this protein is currently thought to be essential for formation of the P-L complex. We recently identified the three relevant phosphate acceptor sites in the VSV Indiana serotype P protein (R. L. Jackson, D. Spadafora, and J. Perrault, Virology 214:189-197, 1995). We now report the effects of substituting Ala at these acceptor sites on transcription reconstitution in vitro and replication of defective interfering virus (DI) templates in vivo. The singly substituted S60A, T62A, and S64A mutants and the doubly substituted S60A/T62A and T62A/S64A mutants, all of which retain some constitutive phosphorylation, were nearly as active as the wild type in both assays. Surprisingly, the nonphosphorylated S60A/S64A protein was also active in transcription (> or = 28%)) and replication (> or = 50%) under optimal conditions. However, this mutant was much less active in in vitro transcription (< or = 5% of wild type) at low P concentrations (<27 nM). In addition, S60A/S64A required higher concentrations of L protein than did the wild type for optimal DI replication in vivo. DI replication efficiency and intracellular accumulation of L, P, and N proteins in the transfected system were very similar to those in VSV-infected cells. We conclude that P protein constitutive phosphorylation is not essential for VSV RNA synthesis per se but likely plays an important role in vivo in facilitating P multimerization and possibly P-L complex formation.

DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1, and TRP-2.

It is believed that DOPA-negative melanocytes in the outer root sheath of the human hair follicle are activated, become identifiable by DOPA staining, and migrate into the epidermis during the repigmenting phase of vitiligo. These cells are difficult to identify, however, and otherwise have not been characterized. These cells are readily identified by immunofluorescence, immunohistochemistry, and immunoelectronmicroscopy using the antibodies NKI/beteb and A4F11, which recognize premelanosome-related antigens. The majority of the outer root sheath melanocytes were found in the mid to the upper portion of the hair follicle. Double staining revealed that these cells were distinct from HLA-DR-bearing dendritic cells. Further immunohistochemical investigation using alpha-PEP-7, alpha-PEP-1, or TMH-1 and alpha-PEP-8 antibodies revealed that outer root sheath melanocytes cannot be identified by antibodies to tyrosinase, TRP-1, or TRP-2, respectively. These cells also did not react with HMB45 antibody, which recognizes a melanosome-associated cytoplasmic antigen. We believe that the inactive outer root sheath melanocytes contain some of the early structural proteins but not any of the enzymatic proteins necessary for melanogenesis. Therefore, activation is the process whereby outer root sheath melanocytes acquire all of the structural and enzymatic proteins necessary for melanogenesis.

Hierarchal constitutive phosphorylation of the vesicular stomatitis virus P protein and lack of effect on P1 to P2 conversion.

In vitro reconstitution of a transcriptionally active VSV polymerase complex (P:L) reportedly requires phosphorylation of the N-terminal domain of P by CKII. Two constitutively phosphorylated sites have been implicated in this activation for both VSV Indiana and New Jersey serotype P proteins. We show here that, in contrast to New Jersey, the Indiana P protein is constitutively phosphorylated on three sites in vivo. The evidence rests on assessing the phosphorylation status of transfected P gene constructs containing all possible combinations of Ala substitutions at Ser60, Thr62, and Ser64. All mutants containing the T62A substitution showed a reduced level of phosphorylation and yielded no P-Thr. Surprisingly the S60A/S64A mutant behaved like the triple substitution and displayed no significant phosphorylation, while the S64A mutant yielded no P-Thr. Phosphorylation of Thr62 therefore depended on prior modification of Ser64. We also tested the ability of our mutant P proteins to convert to the more highly phosphorylated P2 species, a modification essential for transcription in the New Jersey serotype and thought to be carried out by an L-protein-associated kinase. All of our transfected mutant P proteins readily converted to P2 in the presence or absence of L cotransfection, and the latter had no significant effect on P phosphorylation. We conclude that VSV Indiana P protein differs in significant ways from New Jersey P. It is hierarchically and constitutively phosphorylated on a cluster of three sites, not two, suggesting that an additional kinase may be involved. Moreover, Indiana P1 to P2 conversion is independent of prior constitutive phosphorylation and does not require the presence of L protein.

Simultaneous free-tissue transfer and Ilizarov distraction osteosynthesis in lower extremity salvage: case report and review of the literature.

The last decade has witnessed the refinement of microvascular tissue transplantation and its application in treating difficult open wounds. Nonetheless, severe type III tibial injuries continue to challenge reconstructive surgeons. The clinical presentation and surgical treatment of a type IIIB tibial fracture, complicated by long-standing chronic osteomyelitis, angulation deformity, and bone shortening, is presented. This case illustrates the benefit of combining microvascular transplantation of distant tissues with the Ilizarov technique of distraction osteosynthesis in the treatment of complicated lower extremity injuries.

Human hepatic triglyceride lipase expression reduces high density lipoprotein and aortic cholesterol in cholesterol-fed transgenic mice.

We have produced a line of transgenic mice expressing human hepatic triglyceride lipase (hH-TGL) to examine the in vivo effects of hepatic lipase expression on high density lipoprotein catabolism. Activation of metallothionine I promoter-hH-TGL cDNA transgene produced high levels of lipase mRNA in liver, heart, and kidney and elevated enzyme activity as assayed in post-heparin plasma. In a series of hyperlipidemic diet studies in which zinc was included in the diet to induce the transgene, hH-TGL expression was associated with a 34% lowering of plasma HDL-cholesterol levels (p < 0.01) when compared with animals on the same hyperlipidemic diet without zinc. This lowering of HDL cholesterol was paralleled by a decrease in total cholesterol and a decrease in HDL particle size. SDS-polyacrylamide gel electrophoresis analysis of the smaller HDL particles revealed that apolipoprotein AI was still the major apoprotein associated with the HDL. Quantitative analysis of abdominal aortic cholesterol content from the same animals suggests that the observed changes in plasma HDL by hH-TGL over-expression correlated with a decrease in the accumulation of aortic cholesterol (42%, p < 0.01). These data support the hypothesis that hH-TGL mediates a non-receptor pathway for the clearance of cholesterol from the plasma compartment.