Smartphone-based detection of levodopa in human sweat using 3D printed sensors.

Parkinson's disease (PD) is one of the leading neurological disorders negatively impacting health on a global scale. Patients diagnosed with PD require frequent monitoring, prescribed medications, and therapy for extended periods as symptom severity worsens. The primary pharmaceutical treatment for PD patients is levodopa (L-Dopa) which reduces many symptoms experienced by PD patients (e.g., tremors, cognitive ability, motor dysfunction, etc.) through the regulation of dopamine levels in the body. Herein, the first detection of L-Dopa in human sweat using a low-cost 3D printed sensor with a simple and rapid fabrication protocol combined with a portable potentiostat wirelessly connected to a smartphone via Bluetooth is reported. By combining saponification and electrochemical activation into a single protocol, the optimized 3D printed carbon electrodes were able to simultaneously detect uric acid and L-Dopa throughout their biologically relevant ranges. The optimized sensors provided a sensitivity of 83 ± 3 nA/µM from 24 µM to 300 nM L-Dopa. Common physiological interferents found in sweat (e.g., ascorbic acid, glucose, caffeine) showed no influence on the response for L-Dopa. Lastly, a percent recovery of L-Dopa in human sweat using a smartphone-assisted handheld potentiostat resulted in the recovery of 100 ± 8%, confirming the ability of this sensor to accurately detect L-Dopa in sweat.

The effect of medication event reminder monitoring on treatment adherence of TB patients.

BACKGROUND: TB control remains a serious public health problem, compounded by poor treatment adherence, which increases the likelihood of onward transmission. We evaluated the effectiveness of medication event reminder monitoring (MERM) upon treatment adherence in a high TB burden setting.METHODS: We conducted an open-label parallel group randomised controlled trial among pulmonary TB adults. Participants were provided with a MERM device to store their medications. In the intervention arm, the devices were set to provide daily medication intake reminders. Primary outcome was the proportion of patient-months in which at least 6/30 doses were missed. Secondary outcomes included 1) the proportion of patient-months in which at least 14/30 doses were missed, and 2) the proportion of doses missed.RESULTS: Of 2,142 patients screened, 798 (37.3%) met the inclusion criteria and 250 participants were enrolled. The mean ratio (MR) for poor adherence between the intervention and control groups was 0.72 (95% CI 0.55-0.86). The intervention was also associated with a reduction in the proportion of patients missing at least 14/30 doses (MR 0.61, 95% CI 0.54-0.68) and the percentage of total doses missed (MR 0.75, 95% CI 0.68-0.80).CONCLUSION: MERM is effective in improving TB treatment adherence in a resource-limited environment.

3D Printed Ion-Selective Membranes and Their Translation into Point-of-Care Sensors.

This technical note describes a method for fabricating ion-selective membranes (ISMs) for use in potentiometric sensing by using 3D printing technology. Here, we demonstrate the versatility of this approach by fabricating ISMs and investigating their performance in both liquid-contact and solid-contact ion-selective electrode (ISE) configurations. Using 3D printed ISMs resulted in highly stable (drift of ∼17 µV/h) and highly reproducible (<1 mV deviation) measurements. Furthermore, we show the seamless translation of these membranes into reliable, carbon fiber- and paper-based potentiometric sensors for applications at the point-of-care. To highlight the modifiability of this approach, we fabricated sensors for bilirubin, an important biomarker of liver health; benzalkonium, a common preservative used in the pharmaceutical industry; and potassium, an important blood electrolyte. The ability to mass produce sensors using 3D printing is an attractive advantage over conventional methods, while also decreasing the time and cost associated with sensor fabrication.

A comparison of digital chest radiography and Xpert® MTB/RIF in active case finding for tuberculosis.

OBJECTIVE: To compare two community screening tests for TB: sputum examination using Xpert® MTB/RIF and chest radiography (CXR).METHOD: Men aged ≥15 years and women aged >45 years living in 96 sub-communes in Ca Mau, Viet Nam, were invited to provide a single sputum specimen that was tested using Xpert. Participants were also invited to attend a nearby location for digital radiography. Participants whose sputum was Xpert MTB-positive or whose CXR was reported as 'consistent with TB´ were requested to provide two further sputum specimens for culture. The sensitivities of the two tests for detecting TB (defined as sputum culture-positive for Mycobacterium tuberculosis) were compared.RESULTS: There were 72 985 eligible participants, of whom 57 597 (78.9%) participated in Xpert screening, 12 752 (17.5%) had CXR and 11 235 (15.4%) had both tests. We estimated that there were 59 cases of TB, of whom 20 were Xpert MTB-positive (programmatic sensitivity 34.0%) and 47 had CXR reported as 'consistent with TB´ (sensitivity 80.0%, P < 0.0001).CONCLUSION: In community-wide screening for TB, CXR is more sensitive than a single spontaneously expectorated sputum sample tested using Xpert, but it has a substantially lower participation rate.

New neurons use Slit-Robo signaling to migrate through the glial meshwork and approach a lesion for functional regeneration.

After brain injury, neural stem cell-derived neuronal precursors (neuroblasts) in the ventricular-subventricular zone migrate toward the lesion. However, the ability of the mammalian brain to regenerate neuronal circuits for functional recovery is quite limited. Here, using a mouse model for ischemic stroke, we show that neuroblast migration is restricted by reactive astrocytes in and around the lesion. To migrate, the neuroblasts use Slit1-Robo2 signaling to disrupt the actin cytoskeleton in reactive astrocytes at the site of contact. Slit1-overexpressing neuroblasts transplanted into the poststroke brain migrated closer to the lesion than did control neuroblasts. These neuroblasts matured into striatal neurons and efficiently regenerated neuronal circuits, resulting in functional recovery in the poststroke mice. These results suggest that the positioning of new neurons will be critical for functional neuronal regeneration in stem/progenitor cell-based therapies for brain injury.

Clinical predictors of adverse outcome in severe sepsis patients with lactate 2-4 mM admitted to the hospital.

BACKGROUND: Severe sepsis patients with initial lactate level 2-4 mM are commonly considered to have lower risk for mortality and adverse outcomes. AIM: We aim to determine clinical variables that are associated with adverse outcome in these patients. DESIGN: A retrospective cohort study. METHODS: Severe sepsis patients with initial lactate ≥ 2 and < 4 mM admitted to our hospital were examined for any of the following primary outcomes: (i) in-hospital death, (ii) vasopressor requirement, (iii) use of mechanical ventilator, (iv) lactate ≥ 4.0 mM or (v) need care in the intensive care unit (ICU) within 48 h. RESULTS: Five-hundred and thirty-five patients were enrolled, age 58.7 ± 19.3 years, 53.2% male. The most common sources of infection were urinary tract infection and pneumonia, 38.3 and 35.7%, respectively. One-hundred and twenty-four (23.2%) patients had at least one primary adverse outcome within 48 h, including in-hospital death 1.1%, vasopressor requirement 12.9%, use of mechanical ventilator 13.3%, increase lactate ≥ 4.0 mM in 5.6% patients and 21.5% of patients requiring ICU (including 13.8% of the patients admitted directly to ICU from the emergency department, and 7.7% initially admitted to the general medical ward but later required ICU transfer). Altered mentation, hypotension, tachypnea and elevated blood urea nitrogen at admission were associated with the primary outcome in multivariable logistic regression analysis, odds ratio 2.50 (95% confidence interval: 1.54, 4.06), 3.76 (2.31, 6.10), 1.97 (1.22, 3.17) and 1.78 (1.11, 2.83), respectively. CONCLUSIONS: Our study suggests that clinicians should be cautious about the potential adverse outcomes in severe sepsis patients with initial lactate level between 2 and 4 mM and a presentation of altered mentation, hypotension, tachypnea and/or elevated blood urea nitrogen.

Validation of the vascular pedicle width as a diagnostic aid in critically ill patients with pulmonary oedema by novice non-radiology physicians-in-training.

Assessing intravascular volume status in the critically ill patient remains a challenge for intensivists, and the accuracy of such estimation based on bedside examination alone is reported to be nearly a coin toss. In this retrospective study we sought to validate a previously recommended chest radiographic vascular pedicle width (VPW) ≥70 mm for identifying cardiogenic pulmonary oedema (CPO). We additionally assessed whether novice physicians-in-training can reliably measure the VPW. The study included intensive care patients with an existing pulmonary artery catheter. Three independent raters performed measurements of VPW from chest radiographs obtained within three hours of pulmonary artery occlusion pressure measurements. In 80 patients enrolled, a VPW cut-off of ≥70 mm had a 55% sensitivity, 88% specificity, 81% positive predictive value, 69% negative predictive value and 73% accuracy for identifying patients with CPO. Receiver operating characteristic curve analysis showed an area under the curve of 0.72 (95% confidence interval 0.61 to 0.84) for VPW in discriminating CPO from non-cardiogenic pulmonary oedema. Kappa statistics for inter-rater reliability showed Kappa=0.41, 0.42 and 0.85 for each pair of the three raters. In conclusion, the previously accepted VPW cut-off of ≥70 mm is reasonably accurate in discriminating CPO from non-cardiogenic pulmonary oedema. VPW can be measured by physicians-in-training with a comparable performance to previous studies utilising expert radiologists.

LKB1 tumor suppressor regulates AMP kinase/mTOR-independent cell growth and proliferation via the phosphorylation of Yap.

The liver kinase B1 (LKB1) tumor suppressor inhibits cell growth through its regulation of cellular metabolism and apical-basal polarity. The best understood mechanism whereby LKB1 limits cell growth is through activation of the AMP-activated-protein-kinase/mammalian-target-of-rapamycin (AMPK/mTOR) pathway to control metabolism. As LKB1 is also required for polarized epithelial cells to resist hyperplasia, it is anticipated to function through additional mechanisms. Recently, Yes-associated protein (Yap) has emerged as a transcriptional co-activator that modulates tissue homeostasis in response to cell-cell contact. Thus this study examined a possible connection between Yap and LKB1. Restoration of LKB1 expression in HeLa cells, which lack this tumor suppressor, or short-hairpin RNA knockdown of LKB1 in NTERT immortalized keratinocytes, demonstrated that LKB1 promotes Yap phosphorylation, nuclear exclusion and proteasomal degradation. The ability of phosphorylation-defective Yap mutants to rescue LKB1 phenotypes, such as reduced cell proliferation and cell size, suggest that Yap inhibition contributes to LKB1 tumor suppressor function(s). However, failure of Lats1/2 knockdown to suppress LKB1-mediated Yap regulation suggested that LKB1 signals to Yap via a non-canonical pathway. Additionally, LKB1 inhibited Yap independently of either AMPK or mTOR activation. These findings reveal a novel mechanism whereby LKB1 may restrict cancer cell growth via the inhibition of Yap.

Characterization of CrgA, a new partner of the Mycobacterium tuberculosis peptidoglycan polymerization complexes.

The role(s) in cell division of the Mycobacterium tuberculosis Rv0011c gene product, a homolog of the Streptomyces CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-M. tuberculosis CrgA (ECFP-CrgA(MT)) fusion protein is localized to the cell membrane, midcell, and cell pole regions in Mycobacterium smegmatis. Furthermore, the ECFP-CrgA(MT) fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in M. smegmatis. Bacterial two-hybrid assays indicated strong interactions of M. tuberculosis CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgA(MT) was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring. M. tuberculosis cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A M. smegmatis ΔcrgA strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a ΔcrgA strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein.

Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose binding protein (MBP) and a His(8)-tag. Sixty-eight percent of targeted proteins were expressed in high yield (>30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose binding protein as a fusion tag.

Real-space refinement of single-crystal electron diffuse scattering and its application to Bi(2)Ru(2)O(7-δ).

A real-space atomistic refinement approach to the analysis of experimental electron diffraction patterns is described. The method employs the reverse Monte Carlo algorithm to produce atomistic configurations capable of qualitatively reproducing diffuse electron scattering patterns. Its implementation in the program EDRMC is described in detail, together with a number of additional constraints/restraints that can be used to guide the refinement process. In particular, appropriate restraints ensure the individual atomic displacements introduced to model the diffuse scattering patterns are simultaneously consistent with the known average structure. The approach is then used to interpret electron diffraction patterns measured for Bi(2)Ru(2)O(7-δ). The diffuse scattering patterns observed are shown to arise primarily from concerted translations of Bi atoms. These translations can be interpreted in terms of rotations of [O(')Bi(4) ] tetrahedra correlated along the [Formula: see text] crystal axes and uncorrelated along orthogonal directions.

Critical care in the emergency department: A physiologic assessment and outcome evaluation.

OBJECTIVES: The changing landscape of health care in this country has seen an increase in the delivery of care to critically ill patients in the emergency department (ED). However, methodologies to assess care and outcomes similar to those used in the intensive care unit (ICU) are currently lacking in this setting. This study examined the impact of ED intervention on morbidity and mortality using the Acute Physiology and Chronic Health Evaluation (APACHE II), the Simplified Acute Physiology Score (SAPS II), and the Multiple Organ Dysfunction Score (MODS). METHODS: This was a prospective, observational cohort study over a three-month period. Critically ill adult patients presenting to a large urban ED and requiring ICU admission were enrolled. APACHE II, SAPS II, and MODS scores and predicted mortality were obtained at ED admission, ED discharge, and 24, 48, and 72 hours in the ICU. In-hospital mortality was recorded. RESULTS: Eighty-one patients aged 64 +/- 18 years were enrolled during the study period, with a 30.9% in-hospital mortality. The ED length of stay was 5.9 +/- 2.7 hours and the hospital length of stay was 12.2 +/- 16.6 days. Nine (11.1%) patients initially accepted for ICU admission were later admitted to the general ward after ED intervention. Septic shock was the predominant admitting diagnosis. At ED admission, there was a significantly higher APACHE II score in nonsurvivors (23.0 +/- 6.0) vs survivors (19.8 +/- 6.5, p = 0.04), while there was no significant difference in SAPS II or MODS scores. The APACHE II, SAPS II, and MODS scores were significantly lower in survivors than nonsurvivors throughout the hospital stay (p

Role of the CLOCK protein in the mammalian circadian mechanism.

The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, was coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements, a type of transcription factor-binding site, found adjacent to the mouse per1 gene and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.

Prevalence of hepatitis B, hepatitis C, and GB virus C/hepatitis G virus infections in liver disease patients and inhabitants in Ho Chi Minh, Vietnam.

The prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and GB virus C or hepatitis G virus (GBV-C/HGV) infections was determined in 289 patients with liver disease in Ho Chi Minh City and 890 healthy inhabitants of its rural area, Dalat City, Vietnam, respectively. Serum HCV RNA and GBV-C/HGV RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). HBsAg, HCV antibodies, and GBV-C/HGV RNA were detected in 139 (47%), 69 (23%), and ten (3%) subjects, respectively, often accompanied by elevated serum levels of alanine aminotransferase. HBsAg and HCV antibodies or HCV antibodies and GBV-C/HGV RNA were detectable simultaneously in 8% and 2% of the patients, respectively. In the inhabitants, HBsAg, HCV antibodies, and GBV-C/HGV RNA were found in 51 (5.7%), nine (1.0%), and 11 (1.2%) subjects, respectively. Thus, the prevalence of HBsAg, HCV antibodies, and GBV-C/HGV RNA was significantly higher in liver disease patients than those in the general population. In the samples from 69 patients and nine inhabitants who were seropositive for HCV antibodies, HCV RNA was detectable in 42 (61%) and 4 (44%), respectively. In patients with liver disease, ten belonged to HCV genotype 1a, ten to HCV 1b, three to HCV 2a, four to HCV 2b, and two to HCV 3a by PCR with genotype-specific primers. Nine patients had mixed genotypes, and the remaining four were not classified. Of the GBV-C/HGV RNA-positive individuals, two patients and two inhabitants were positive for HBsAg, while none of the residents had HCV antibodies, although six HCV antibodies (60%) and four HCV RNA (40%) were found in patients. When a phylogenetic tree of GBV-C/HGV was constructed based on the nucleotide sequences, the 21 isolates were classified into at least two genotypes; four isolates belonged to G2, and 17 to G3. The results indicate that in Ho Chi Minh HCV infection prevails with broad distribution of genotypes together with HBV infection among patients with liver disease. This study suggests that GBV-C/HGV infection occurs independently in the two different districts in association with HCV infection.

Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblasts.

Mutations in the fibroblast growth factor receptor (FGFR) gene family recently have been shown to underlie several hereditary disorders of bone development, with specific FGFR3 mutations causing achondroplasia (Ach) and thanatophoric dysplasia (TD). However, for none of these mutations has the defect in receptor function been demonstrated directly and, therefore, for none has the pathophysiological mechanism of the disease been defined. Using our established techniques for single-cell ratiometric real-time calcium image analysis, we defined the nature of the basic fibroblast growth factor (bFGF)-induced calcium signal in human diploid fibroblasts, and, in blinded studies, have analyzed the bFGF-induced signals from 18 independent fibroblast cell lines, including multiple lines from patients with known mutant alleles of FGFR3 and syndromes of Ach or TD. Control cells responded with transient increases in intracellular calcium, with many cells showing oscillatory calcium waves. Homozygous Ach cell lines failed to signal, whereas heterozygous Ach lines responded nearly normally. We observed heterogeneous signals in TD heterozygotes: the unresponsive lines all turned out to carry TD1 alleles, whereas all responsive lines had TD2 alleles. Since FGFR1, 2 and 3 receptors are known to be expressed in fibroblasts, our results suggest that specific mutant FGFR3 alleles can function in a dosage-dependent dominant-negative fashion to inactivate FGFR signaling.

Calcium is permeable through a maitotoxin-activated nonselective cation channel in mouse L cells.

The shellfish poison maitotoxin causes the irreversible opening of nonselective cation channels in mouse L cell fibroblasts, consistent with the action of this toxin in other cell types and the previously demonstrated existence of 28-pS voltage-insensitive nonselected cation channels that are activated by platelet-derived growth factor in these cells. Toxin-induced opening of these nonselective cation channels led to increases of intracellular calcium and secondary activation of calcium-activated potassium channel. These effects were completely dependent on influx of extracellular calcium, supporting the conclusion that the maitotoxin-activated nonselective cation channels are permeable to calcium as well as to sodium and potassium. The implication of this finding is that calcium signaling through this channel underlies its links into the growth factor response.

Diaspirin cross-linked hemoglobin is efficacious in gut resuscitation as measured by a GI tract optode.

The objective of this study was to compare the efficacy of diaspirin cross-linked hemoglobin (DCLHb) with that of standard resuscitative fluids in restoring intestinal mucosal oxygenation and villous architecture after hemorrhage. Male rats were bled to a base deficit of 5 +/- 2 nmol/l under propofol anesthesia and monitored for 90 minutes postresuscitation with DCLHb, blood, lactated Ringer's solution, albumin, or nothing (DNR) for mucosal oxygen tension (Pmo2) and physiologic and laboratory parameters. Small intestinal histologic specimens were obtained and scored independently by two investigators blinded to therapy on a scale of 0 (normal) to 4 (worst). All treatments restored Pmo2; only DCLHb did so without exceeding baseline values. For untreated rats (DNR), Pmo2 was not restored. Normal mucosal architecture was maintained only in DCLHb-treated rats. As Pmo2 increased, mucosal score improved. In a rat model of controlled hemorrhage, Pmo2 changes measured by an optode correlated with gut histological abnormalities. By these criteria, DCLHb is superior to crystalloid, colloid, and blood in gut resuscitation.

Effect of photoperiod on the size of the Leydig cell population and the rate of recruitment of Leydig cells in adult Syrian hamsters.

The number of Leydig cells was determined by stereologic procedures in adult Syrian hamsters housed in long days (14L:10D) to maintain testicular activity (active), in short days (5L:19D) for 12-13 wk to induce testicular regression (photoperiod-induced regressed), or in short days for a period of 21 wk or more to allow spontaneous gonadal recrudescence (spontaneously recrudesced). Testes were removed, sliced, fixed, embedded in Epon 812, and observed by bright-field microscopy. Testicular and seminal vesicle weights, plasma testosterone concentration, total Leydig cell volume per testis, and volume of single Leydig cell were greater (p less than 0.01) in active and recrudesced animals than in regressed animals. The density of Leydig cells was greater in the regressed testes, but the total number per testis was not influenced by photoperiod. In Experiment 2, the rate of recruitment of Leydig cells was determined in 5 adult hamsters exposed to long days (active) or 5 hamsters whose testes were regressed by exposure of animals to short days for 13 wk followed by long-day exposure to initiate testicular growth (photoperiod-induced recrudescing). Hamsters were injected for 3 days/wk for 3 wk with tritiated thymidine, 0.5 or 1 microCi/g body weight. Testes were fixed and tissues prepared, as above, and processed for autoradiography. Again, the photoperiod did not influence the number of Leydig cells per testis. Labeling of Leydig cell nuclei revealed that recruitment of new Leydig cells occurred at approximately 1.3% per day in recrudescing testes but also occurred at approximately 0.6% per day in active testes. Without change in the total number of Leydig cells, new Leydig cells were added continually to the existing population in adult hamsters with either recrudescing or active testes.