Integrated Microfluidic System for Rapid DNA Fingerprint Analysis: A Miniaturized Integrated DNA Analysis System (MiDAS)-Swab Sample-In to DNA Profile-Out.

A fully automated rapid DNA analysis system requires integrating several operational elements performing multiple steps onto one single microfluidic platform. The functions to include on the microfluidic platform consist of substrate lysis, lysate DNA extraction, single or multiplexed PCR amplification, amplicon separation, and product readout. Here we describe a fully automated integrated system for forensic short tandem repeat (STR) analysis of reference samples, achieving buccal swab-in and DNA profile-out.

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

Gait and balance in treatment-naïve active alcoholics with and without a lifetime drug codependence.

BACKGROUND: Disturbed gait and balance are among the most consistent sequelae of chronic alcoholism. However, although a majority of alcoholics have never sought treatment, most investigations showing ataxia in alcohol-dependent individuals have relied on samples drawn from treated populations. In addition, few studies have addressed the associations of codependence on other drugs with alcoholic gait and balance disturbance. METHODS: This study employed the Walk-a-Line Ataxia Battery (Fregly et al. Alcohol Clin Exp Res 1972;43:395-399) to assess gait and balance in treatment-naïve, actively drinking alcohol-dependent men and women (TNA; n = 69) who were dependent on alcohol only (ALC; n = 43), or who also had a lifetime drug dependence (ALC + DRG; n = 26; i.e., methamphetamine, cocaine, opiates, and/or marijuana), compared with nonsubstance abusing controls (NSAC; n = 74).We also examined associations between lifetime alcohol use and age with gait and balance measures. RESULTS: Our main findings were (i) no evidence of disturbed gait and balance in ALC versus NSAC and (ii) significantly disturbed gait and balance in ALC + DRG, relative to both NSAC and ALC, along with steeper age-associated decline in gait and balance performance in ALC versus ALC + DRG. CONCLUSIONS: Our results provide evidence consistent with previous studies that TNA (without a lifetime drug codependence) may represent a population that is different and less impaired (including in gait and balance) than treated alcoholics. Additionally, we provide evidence that ALC + DRG, with greater alcohol use and family drinking density than ALC, have an accelerated effect of age on gait and balance disturbance compared with both NSAC and ALC. The ALC + DRG group likely represents a subset of TNA with different characteristics than ALC.

Persistent but less severe ataxia in long-term versus short-term abstinent alcoholic men and women: a cross-sectional analysis.

BACKGROUND: Disturbed gait and balance are among the most consistent and salient sequelae of chronic alcoholism. Results of small sample longitudinal investigations have provided evidence that partial recovery of gait and balance functions in alcoholics may be achieved with abstinence. However, abstinence durations reported have been limited, and their power and generalizability have suffered from small sample sizes. METHODS: In this study, we employed a cross-sectional approach to assess gait and balance functions in short-term (6 to 15 weeks) abstinent alcoholics (STAA; n = 70) and long-term (minimum 18 months, mean = 7.38 years) abstinent alcoholics (LTAA; n = 82). STAA and LTAA did not differ with respect to lifetime alcohol consumption, family drinking density, or years of education. In addition, we examined the effects of gender and alcohol use variables. RESULTS: Our main findings were: (i) persistent disturbed gait and balance in STAA and disturbed standing balance in LTAA; (ii) overall less impaired performance of LTAA compared with STAA on gait and balance measures; and (iii) worse performance of STAA (but not LTAA) women, compared with men, on standing balance without visual control. CONCLUSIONS: Our results suggest that alcoholics' gait and balance can continue to recover with long abstinence from alcohol, but that deficits persist, especially in eyes-closed standing balance. In addition, our results are consistent with more severe alcohol-induced ataxia in women than in men but suggest that with extended abstinence, women recover gait and balance function to a level comparable with men.

Subcortical volumes in long-term abstinent alcoholics: associations with psychiatric comorbidity.

BACKGROUND: Research in chronic alcoholics on memory, decision-making, learning, stress, and reward circuitry has increasingly highlighted the importance of subcortical brain structures. In addition, epidemiological studies have established the pervasiveness of co-occurring psychiatric diagnoses in alcoholism. Subcortical structures have been implicated in externalizing pathology, including alcohol dependence, and in dysregulated stress and reward circuitry in anxiety and mood disorders and alcohol dependence. Most studies have focused on active or recently detoxified alcoholics, while subcortical structures in long-term abstinent alcoholics (LTAA) have remained relatively uninvestigated. METHODS: Structural MRI was used to compare volumes of 8 subcortical structures (lateral ventricles, thalamus, caudate, putamen, pallidum, hippocampus, amygdala, and nucleus accumbens) in 24 female and 28 male LTAA (mean abstinence=6.3 years, mean age= 46.6 years) and 23 female and 25 male nonalcoholic controls (NAC) (mean age=45.6 years) to explore relations between subcortical brain volumes and alcohol use measures in LTAA and relations between subcortical volumes and psychiatric diagnoses and symptom counts in LTAA and NAC. RESULTS: We found minimal differences between LTAA and NAC in subcortical volumes. However, in LTAA, but not NAC, volumes of targeted subcortical structures were smaller in individuals with versus without comorbid lifetime or current psychiatric diagnoses, independent of lifetime alcohol consumption. CONCLUSIONS: Our finding of minimal differences in subcortical volumes between LTAA and NAC is consistent with LTAA never having had volume deficits in these regions. However, given that imaging studies have frequently reported smaller subcortical volumes in active and recently detoxified alcoholics compared to controls, our results are also consistent with the recovery of subcortical volumes with sustained abstinence. The finding of persistent smaller subcortical volumes in LTAA, but not NAC, with comorbid psychiatric diagnoses, suggests that the smaller volumes are a result of the combined effects of chronic alcohol dependence and psychiatric morbidity and suggests that a comorbid psychiatric disorder (even if not current) interferes with the recovery of subcortical volumes.

Cognitive performance in treatment-naïve active alcoholics.

BACKGROUND: Most studies reporting cognitive deficits in chronic alcoholics have relied on treatment samples (predominantly men) from inpatient or outpatient treatment facilities. However, the majority of chronic alcoholics have never been in treatment and there is increasing evidence that treated and non-treatment-seeking alcoholic samples come from different populations with regard to alcohol use and other factors related to the severity of disease. Accordingly, in the present study, we assessed a broad range of cognitive functions in 55 treatment-naïve alcohol-dependent (TNAD) individuals and 55 nonalcoholic controls (NAC) matched for age and education. In addition, a goal of the present study was to assess potential differential effects of alcohol dependence on cognitive performance in TNAD men and women. METHODS: Comprehensive neuropsychological assessment was conducted on TNAD and NAC. The following 9 performance domains, each consisting of multiple measures, were examined: attention, auditory working memory, verbal processing, abstraction/cognitive flexibility, psychomotor function, immediate memory, delayed memory, reaction time, and spatial processing. RESULTS: Analysis revealed no cognitive deficits in TNAD, relative to NAC, in any of the 9 cognitive domains. TNAD performed better than NAC in the attention domain. In addition, while men performed better than women in the spatial domain, there were no TNAD versus NAC group by gender interactions for any domain. CONCLUSIONS: Our results extend findings that TNAD show minimal behavioral effects of chronic heavy alcohol use and are consistent with the contention that TNAD are relatively cognitively intact. Differences between our findings and those often reported for alcoholics recruited from treatment settings may be understood in terms of differences in alcohol use, along with genetic, psychiatric, and nutritional factors. In addition, the lack of differential effects of alcohol dependence on male and female cognitive performance in our study suggests that TNAD men and women do not differ in the severity of cerebral consequences of alcohol dependence.

Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile.

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.

Cyclic 3', 5' guanosine monophosphate and larval midgut ecdysone 20-monooxygenase activity of the tobacco hornworm, Manduca sexta.

During the last larval stadium of the tobacco hornworm, Manduca sexta, midgut ecdysone 20-monooxygenase (E20M) activity increases 50-fold coincident with wandering onset. In our study, the effects of the ecdysone agonist RH-5849 and second messengers, dibutyryl cyclic 3', 5' adenosine monophosphate (dibutyryl cAMP) and dibutyryl cyclic 3', 5' guanosine monophosphate (dibutyryl cGMP), either alone or combined with other additives, on midgut E20M were assessed in vitro and in vivo. Pre-incubation of midguts with dibutyryl cAMP was ineffective, but dibutyryl cGMP elevated E20M activity. In vivo, the 50-fold increase in E20M activity was diminished by injections of guanylate cyclase inhibitors alone or combined with RH-5849. Guanylate cyclase inhibitor effects were significantly diminished when co-injected with dibutyryl cGMP, or a guanylate cyclase activator. Although dibutyryl cGMP injections into head- or thorax-ligated pre-wandering larvae did not elevate midgut E20M activity, they did synergize when co-injected with a suboptimal dose of RH-5849, thereby eliciting significant activity increases. Radioimmunoassays of midgut cGMP content throughout the M. sexta fifth larval instar revealed that levels of cGMP significantly increased between days four and five consistent with the 50-fold E20M activity increase. These data indicate cGMP involvement in the ecdysone-mediated increase in midgut E20M activity.

Feasibility of Internet training for care staff of residents with dementia: the CARES program.

This study evaluated an Internet-based training module, CARES(®) (Connect with the resident; Assess behavior; Respond appropriately; Evaluate what works; Share with the team), to determine its feasibility for certified nursing assistants (CNAs) in three nursing homes and one assisted living facility. Pre- and posttest questionnaires were administered to 40 CNAs to determine improvements in dementia care knowledge and perceptions of competence in dementia care. Dementia care knowledge improved significantly after CARES training. More than 85% of the sample agreed or strongly agreed that the CARES protocol improved mastery, improved care competency, and reduced stress related to care of residents with dementia. Open-ended feedback indicated that CARES provided CNAs with new information and skills pertaining to dementia care. The results suggest that Internet-based programs such as CARES represent time- and cost-efficient methods to deliver dementia care training in long-term care settings.

Midgut and fatbody mitochondrial transhydrogenase activities during larval-pupal development of the tobacco hornworm, Manduca sexta.

Midgut and fatbody mitochondria from fifth larval instar Manduca sexta display a membrane-associated transhydrogenase that catalyzes a reversible hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming activity occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg(++)-dependent ATPase. During the ten-day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the fifty-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations were made apparent. Moreover, that the increases in all transhydrogenase activities resulted from de novo enzyme synthesis were indicated by the cycloheximide-dependent reductions in these activities.

Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta.

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.

Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity.

The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M.