Prevalence of non-convulsive seizure and other electroencephalographic abnormalities in ED patients with altered mental status.

UNLABELLED: Four to ten percent of patients evaluated in emergency departments (ED) present with altered mental status (AMS). The prevalence of non-convulsive seizure (NCS) and other electroencephalographic (EEG) abnormalities in this population is unknown. OBJECTIVES: To identify the prevalence of NCS and other EEG abnormalities in ED patients with AMS. METHODS: A prospective observational study at 2 urban ED. Inclusion: patients ≥13 years old with AMS. Exclusion: An easily correctable cause of AMS (e.g. hypoglycemia). A 30-minute standard 21-electrode EEG was performed on each subject upon presentation. OUTCOME: prevalence of EEG abnormalities interpreted by a board-certified epileptologist. EEGs were later reviewed by 2 blinded epileptologists. Inter-rater agreement (IRA) of the blinded EEG interpretations is summarized with κ. A multiple logistic regression model was constructed to identify variables that could predict the outcome. RESULTS: Two hundred fifty-nine patients were enrolled (median age: 60, 54% female). Overall, 202/259 of EEGs were interpreted as abnormal (78%, 95% confidence interval [CI], 73-83%). The most common abnormality was background slowing (58%, 95% CI, 52-68%) indicating underlying encephalopathy. NCS (including non-convulsive status epilepticus [NCSE]) was detected in 5% (95% CI, 3-8%) of patients. The regression analysis predicting EEG abnormality showed a highly significant effect of age (P < .001, adjusted odds ratio 1.66 [95% CI, 1.36-2.02] per 10-year age increment). IRA for EEG interpretations was modest (κ: 0.45, 95% CI, 0.36-0.54). CONCLUSIONS: The prevalence of EEG abnormalities in ED patients with undifferentiated AMS is significant. ED physicians should consider EEG in the evaluation of patients with AMS and a high suspicion of NCS/NCSE.

A randomized controlled trial of surface neuromuscular electrical stimulation applied early after acute stroke: effects on wrist pain, spasticity and contractures.

OBJECTIVES: To investigate effects of surface neuromuscular electrical stimulation applied early after stroke to the wrist and finger extensor muscles on upper limb pain, spasticity and contractures in patients with no functional arm movement. DESIGN: Secondary analysis from a Phase II, randomized, controlled, single-blind study. SETTING: An acute hospital stroke unit. SUBJECTS: Patients with no useful arm function within six weeks of a first stroke. INTERVENTION: Patients were randomized to treatment (30-minute sessions of surface neuromuscular stimulation to wrist and finger extensors and 45 minutes of physiotherapy) or control (45 minutes of physiotherapy) groups. All patients had access to routine care. Treatment was given for six weeks from recruitment. RESULTS: Ninety patients (49% male, median age 74 years (range 32-98), median time since stroke onset three weeks (range one to six weeks)) were included. Treatment compliance was variable (mean 28%). The treatment prevented the development of pain (mean difference in rate of change 0.4 units/week, 95% confidence interval (CI) 0.09 to 0.6). Treatment may have prevented a deterioration in contractures (quantified by measuring passive range of movement) in severely disabled patients (mean rate of deterioration -0.5 deg/week; 95% CI -0.9 to -0.06). There were no significant changes in stiffness and spasticity. CONCLUSION: Surface neuromuscular electrical stimulation reduces pain in stroke patients with a non-functional arm. There was some evidence that treatment with electrical stimulation was beneficial in reducing contractures. Treatment had no effect on spasticity.

The accuracy of the olfactory sense in detecting alcohol intoxication in trauma patients.

INTRODUCTION: A common presentation to the emergency department (ED) is the trauma patient with altered sensorium who is presumed to be alcohol intoxicated by physicians based on their olfactory sense. ED physicians may often leave patients suspected of alcohol intoxication aside until the effects wear off, potentially missing trauma as the source of confusion. This often results in delays in diagnosing acute potentially life-threatening injuries in patients with presumed alcohol intoxication. OBJECTIVE: This study aimed to determine the accuracy of a physician's olfactory sense for diagnosing alcohol intoxication. METHODS: Patients suspected of major trauma in the ED underwent an evaluation by the examining physician for alcohol odour and a blood alcohol level. Alcohol intoxication was defined as a serum ethanol level ≥80 mg/100 ml. Data were reported as means with 95% CI or proportions with IQR 25-75%. RESULTS: 151 patients (70% men) were enrolled, with a median age of 45 years (IQR 33-56). The prevalence of alcohol intoxication was 43% (95% CI 35% to 51%). OPERATING CHARACTERISTICS: Physician assessment of alcohol intoxication: sensitivity 84% (95% CI 73% to 92%), specificity 87% (95% CI 78% to 93%), positive likelihood ratio 6.6 (95% CI 3.8 to 11.6), negative likelihood ratio 0.18 (95% CI 0.1 to 0.3) and accuracy 86% (95% CI 80% to 91%). 7.3% (95% CI 4% to 13%) of patients were falsely suspected of being intoxicated. CONCLUSIONS: Although the physicians had a high degree of accuracy in identifying patients with alcohol intoxication based on their olfactory sense, they still falsely overestimated intoxication in significant numbers of non-intoxicated trauma patients.

The challenge of recruiting in primary care for a trial of telemonitoring in asthma: an observational study.

AIM: Achieving target recruitment in randomized controlled trials (RCTs) is challenging. This paper compares our experience of recruiting for an RCT with the predictions made in our proposal. METHODS: Participating UK primary care practices searched their computer databases to identify patients (12 years and over) with asthma who may be poorly controlled. Postal invitations were sent to all patients identified. Respondees were prescreened by phone, to assess their asthma control and establish their mobile phone suitability. Potentially eligible patients were booked for a trial recruitment visit. RESULTS: We recruited 288 patients (2.4% of those invited) across 32 practices, with a total list size of 311,926 patients. This compares to our predicted recruitment of 312 patients from a population of 72,000 patients in six to eight practices. In addition to the recognized problem of poor response rates, the major challenges were insufficiently discriminating computer searches and incompatibilities between mobile phone handsets, networks and the asthma application. CONCLUSION: Our data have implications for clinicians, managers, and researchers in primary care. Researchers in this area may wish to consider our data when designing their recruitment strategies. Improved coding of asthma morbidity data in clinical practice would ease identification of poorly controlled patients, both for clinical interventions and recruitment to trials. If telehealth is to become mainstream, there needs to be standardization of applications, operating platforms, and network capabilities.

Complete genome sequence of phytopathogenic Pectobacterium carotovorum subsp. carotovorum bacteriophage PP1.

Pectobacterium carotovorum subsp. carotovorum is a phytopathogen causing soft rot disease on diverse plant species. To control this plant pathogen, P. carotovorum subsp. carotovorum-targeting bacteriophage PP1 was isolated and its genome was completely sequenced to develop a novel biocontrol agent. Interestingly, the 44,400-bp genome sequence does not encode any gene involved in the formation of lysogen, suggesting that this phage may be very useful as a biocontrol agent because it does not make lysogen after host infection. This is the first report on the complete genome sequence of the P. carotovorum subsp. carotovorum-targeting bacteriophage, and it will enhance our understanding of the interaction between phytopathogens and their targeting bacteriophages.

Can surface neuromuscular electrical stimulation of the wrist and hand combined with routine therapy facilitate recovery of arm function in patients with stroke?

OBJECTIVE: To investigate whether treatment with surface neuromuscular electrical stimulation to the wrist extensors improves recovery of arm function in severely disabled patients with stroke. DESIGN: Single blinded randomized controlled trial. SETTING: Acute stroke unit and stroke rehabilitation wards of a university hospital. PARTICIPANTS: Patients with no upper limb function (Action Research Arm Test [ARAT] score 0) (N=90; mean age ± SD, 74±11y; 49% men) were recruited to the study within 6 weeks of stroke. Only 67 participants were alive at the end of the study and data from 66 of these people were analyzed. INTERVENTIONS: Participants were randomized to surface neuromuscular electrical stimulation using surface electrical stimulators for 30 minutes, twice in a working day for 6 weeks in addition to standardized upper limb therapy or just standardized upper limb therapy. MAIN OUTCOME MEASURE: The primary outcome measure was the ARAT score. Assessments were made at baseline and at 6, 12, 24, and 36 weeks after recruitment. RESULTS: There were statistically significant improvements in measures of wrist extensor (mean difference 0.5; 95% confidence interval [CI], 0.0-1.0) and grip strength (mean difference 0.9; 95% CI, 0.1-1.7) over the treatment period. Arm function (ARAT score) was not significantly different between the groups over the treatment period at 6 weeks (mean difference 1.9; 95% CI, -2.9 to 6.8) or over the study period at 36 weeks (mean difference 6.4; 95% CI, -1.8 to 14.7), and the rate of recovery was not significantly different (mean difference 0.7; 95% CI, -0.2 to 1.6). CONCLUSIONS: In patients with severe stroke, with no functional arm movement, electrical stimulation of wrist extensors improves muscle strength for wrist extension and grip, and larger studies are required to study its influence on arm function.

Clinical and cost effectiveness of mobile phone supported self monitoring of asthma: multicentre randomised controlled trial.

OBJECTIVE: To determine whether mobile phone based monitoring improves asthma control compared with standard paper based monitoring strategies. DESIGN: Multicentre randomised controlled trial with cost effectiveness analysis. SETTING: UK primary care. PARTICIPANTS: 288 adolescents and adults with poorly controlled asthma (asthma control questionnaire (ACQ) score ≥ 1.5) from 32 practices. INTERVENTION: Participants were centrally randomised to twice daily recording and mobile phone based transmission of symptoms, drug use, and peak flow with immediate feedback prompting action according to an agreed plan or paper based monitoring. MAIN OUTCOME MEASURES: Changes in scores on asthma control questionnaire and self efficacy (knowledge, attitude, and self efficacy asthma questionnaire (KASE-AQ)) at six months after randomisation. Assessment of outcomes was blinded. Analysis was on an intention to treat basis. RESULTS: There was no significant difference in the change in asthma control or self efficacy between the two groups (ACQ: mean change 0.75 in mobile group v 0.73 in paper group, mean difference in change -0.02 (95% confidence interval -0.23 to 0.19); KASE-AQ score: mean change -4.4 v -2.4, mean difference 2.0 (-0.3 to 4.2)). The numbers of patients who had acute exacerbations, steroid courses, and unscheduled consultations were similar in both groups, with similar healthcare costs. Overall, the mobile phone service was more expensive because of the expenses of telemonitoring. CONCLUSIONS: Mobile technology does not improve asthma control or increase self efficacy compared with paper based monitoring when both groups received clinical care to guidelines standards. The mobile technology was not cost effective. TRIAL REGISTRATION: Clinical Trials NCT00512837.

Prophylactic antibiotics for simple hand lacerations: time for a clinical trial?

BACKGROUND: Simple hand lacerations (not involving bones, tendons, nerves, or vessels) are a common emergency department (ED) complaint. Whilst the practices of irrigation, debridement, foreign body removal, and suture repair are well accepted, the use of prophylactic antibiotics is not. Without evidenced-based guidelines, practice is left to physician preference. OBJECTIVES: The aim of this study was to assess the need for, and the feasibility to perform, a randomised controlled trial to evaluate the role of prophylactic antibiotics in simple hand lacerations. METHODS: The study was done in three phases: (1) estimation of the national ED burden of simple hand lacerations and the use of antibiotic prophylaxis; (2) assessment of indications for antibiotic prophylaxis and (3) investigation of patient willingness to enrol in a randomised controlled trial and their preferred outcomes from simple hand lacerations. For Phase 1, we analysed the 2007 National Hospital Ambulatory Medical Care Survey. For Phase 2, we surveyed ED physicians in three urban teaching institutions (two in Brooklyn, NY and one in Washington, DC). For Phase 3, we surveyed ED patients at the same three institutions. RESULTS: Phase 1: out of 116.8 million ED visits nationally in 2007, 1.8 million (1.6%) were due to simple hand lacerations, of which 1.3 million (71%) required repair. Of those repaired, 27% (95% CI, 19-35%) were prescribed prophylactic antibiotics, most commonly cephalexin (73%). Phase 2: out of 108 providers surveyed, 69 (64%) responded. 16% (95% CI, 9-27%) reported prescribing prophylactic antibiotics routinely, most commonly cephalexin (84%, 95% CI, 67-93%). The degree of contamination was the most important factor (91%, 95% CI, 82-96%) in the physicians' decision to prescribe antibiotics. Phase 3: of the 490 patients surveyed, 64% (95% CI, 60-68%) expressed interest in participating in a study to evaluate the use of prophylactic antibiotics. Their primary concern was prevention of infection (77%, 95% CI, 73-81%). CONCLUSION: Simple hand lacerations represent a substantial number of ED visits in the United States. Absence of clear guidelines, disparity in physician practice, and patient interest in infection prevention all support performing a prospective randomised controlled trial to establish the role of antibiotic prophylaxis in simple hand lacerations.

Nonconvulsive seizures in patients presenting with altered mental status: an evidence-based review.

Definitive diagnosis of nonconvulsive seizures (NCS) can be made only by electroencephalography, and delay in diagnosis can increase morbidity, resource utilization, and length of hospitalization. We performed an evidence-based literature review to estimate the prevalence of NCS in patients with altered mental status (AMS) of unknown cause. PUBMED, EMBASE, the Cochrane Library, and other resources were searched for studies that included AMS and seizure as topics. The resulting 276 articles were screened for predetermined inclusion and exclusion criteria, leaving 5 studies enrolling 478 patients for review. The prevalence of NCS in patients with AMS ranged from 8 to 30% (overall prevalence of 21.5%, 95% CI: 18-25%), suggesting that the prevalence of NCS is sufficiently high to consider routine use of urgent electroencephalography in such patients. However, methodological weaknesses limit the generalizability of the results. A large, prospective study enrolling and screening for NCS in all patients who present with acute AMS is needed.

Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

A new esterase EstD2 isolated from plant rhizosphere soil metagenome.

Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K(m) and K(cat) values were determined to be 79.4 µM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.

Biochemistry of microbial degradation of hexachlorocyclohexane and prospects for bioremediation.

Lindane, the gamma-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and alpha-, beta-, and delta-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.

An investigation into the agreement between clinical, biomechanical and neurophysiological measures of spasticity.

OBJECTIVE: To quantify agreement between three clinically usable methods of measuring spasticity. METHODS: Patients with a first stroke who had no useful functional movement in the upper limb within six weeks from stroke onset were eligible to participate. Spasticity at the wrist joint was simultaneously measured using three methods, during an externally imposed passive stretch at two (uncontrolled) displacement velocities. The measures used were a common clinical measure (modified Ashworth Scale), a biomechanical measure (resistance to passive movement) and a neurophysiological measure (muscle activity). RESULTS: One hundred patients (54 men and 46 women) with a median age of 74 years (range 43-91) participated. Median time since stroke was three weeks (range 1-6), the right side was affected in 52 patients and the left in 48 patients. Based on muscle activity measurement, 87 patients had spasticity. According to the modified Ashworth score 44 patients had spasticity. Sensitivity of modified Ashworth score, when compared with muscle activity recordings, was 0.5 and specificity was 0.92. Based on muscle activity patterns, patients could be classified into five subgroups. The biomechanical measures showed no consistent relationship with the other measures. CONCLUSION: The presentations of spasticity are variable and are not always consistent with existing definitions. Existing clinical scales that depend on the quantification of muscle tone may lack the sensitivity to quantify the abnormal muscle activation and stiffness associated with common definitions of spasticity. Neurophysiological measures may provide more clinically useful information for the management and assessment of spasticity.

Nuclear factor-kappa B signaling in skeletal muscle atrophy.

Skeletal muscle atrophy/wasting is a serious complication of a wide range of diseases and conditions such as aging, disuse, AIDS, chronic obstructive pulmonary disease, space travel, muscular dystrophy, chronic heart failure, sepsis, and cancer. Emerging evidence suggests that nuclear factor-kappa B (NF-kappaB) is one of the most important signaling pathways linked to the loss of skeletal muscle mass in various physiological and pathophysiological conditions. Activation of NF-kappaB in skeletal muscle leads to degradation of specific muscle proteins, induces inflammation and fibrosis, and blocks the regeneration of myofibers after injury/atrophy. Recent studies employing genetic mouse models have provided strong evidence that NF-kappaB can serve as an important molecular target for the prevention of skeletal muscle loss. In this article, we have outlined the current understanding regarding the role of NF-kappaB in skeletal muscle with particular reference to different models of muscle wasting and the development of novel therapy.

Nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica and construction of a conjugative shuttle vector.

The complete nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica DSM 43387 was determined. Sequence analysis revealed that pA387 is 30,157 bp long and has a G+C content of 71.74%. To obtain a minimal transferable replicon capable of self-replication, a 2,176 bp fragment of pA387 was cloned, and we demonstrated that this fragment is sufficient for autonomous replication. The replication region of pA387 exhibited no significant homology to any known replication proteins available in databases. Putative maintenance and transfer functions were identified on pA387. The predicted products of open reading frames, ORF 2 and ORF 12, resembled the plasmid stabilizing proteins, a DNA resolvase and a ParA protein, respectively. The putative translational products of ORF 15 and ORF 16 showed similarity to known bacterial conjugation proteins, TraG and TraA, respectively. A conjugative Escherichia coli -Amycolatopsis shuttle-cloning vector was constructed by using the pA387 replicon and designated pSETRL1. Shuttle vector pSETRL1 successfully transformed Amycolatopsis mediterranei DSM 40773 and Amycolatopsis orientalis NBRC 12806 by conjugation and electroporation, and is likely to be a useful vector in Amycolatopsis research.

From bacterial genomics to metagenomics: concept, tools and recent advances.

In the last 20 years, the applications of genomics tools have completely transformed the field of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, first describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting findings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world.

Localization of HCH catabolic genes (lin genes) in Sphingobium indicum B90A.

The locations of hexachlorocyclohexane (HCH) catabolic (lin) genes were investigated in HCH degrading sphingomonad, Sphingobium indicum B90A (that was isolated from India). Southern blot analysis revealed the presence of linA1, linC, linDER and linX (linX1 and linX2) on the plasmid DNA in Sphingobium indicum B90A.

Haloalkane dehalogenase LinB is responsible for beta- and delta-hexachlorocyclohexane transformation in Sphingobium indicum B90A.

Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform beta- and delta-hexachlorocyclohexane (beta- and delta-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with beta- and delta-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including beta-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade beta- and delta-HCH.

Diversity, distribution and divergence of lin genes in hexachlorocyclohexane-degrading sphingomonads.

Two forms of hexachlorocyclohexane (HCH), gamma-HCH (lindane) and technical HCH (incorporating alpha-, beta-, gamma- and delta- isomers), have been used against agricultural pests and in health programs since the 1940s. Although all the isomers are present in the milieu, delta- and beta-HCH isomers are the most problematic and present a serious environmental problem. Bacteria that degrade HCH isomers have been isolated from HCH contaminated soils from different geographical locations around the world (from the family Sphingomonadaceae). Interestingly, all these bacteria contain nearly identical lin genes (responsible for HCH degradation), which are diverging to perform several catabolic functions. The organization and diversity of lin genes have been studied among several sphingomonads, and they have been found to be associated with plasmids and IS6100, both of which appear to have a significant role in their horizontal transfer. The knowledge of the molecular genetics, diversity and distribution of lin genes, and the potential of sphingomonads to degrade HCH isomers, can now be used for developing bioremediation techniques for the decontamination of HCH contaminated sites.

Development of cloning vectors and transformation methods for Amycolatopsis.

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA- rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.