Classification of formalin-fixed bladder cancer cells with laser tweezer Raman spectroscopy.

Bladder cancer is a common cancer that is relatively hard to detect at an early stage because of its non-obvious symptoms. It is known that bladder cells can be found in urine samples which potentially could be used for early detection of bladder cancer. Raman spectroscopy is a powerful non-invasive tool for accessing biochemical information of cells. Combined with laser tweezers, to allow isolation of single cells, Raman spectroscopy has been used to characterise a number of bladder cells that might be found in a urine sample. Using principal component-canonical variates analysis (PC-CVA) and k-fold validation, the results shows that the invasive bladder cancer cells can be identified with accuracy greater than 87%. This demonstrates the potential of developing an early detection method that identifies the invasive bladder cancer cells in urine samples.

Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola.

The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes "alarmone-like" nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.

Reducing Specimen Rejection Rates Using Concentration-Dependent Hemolysis Rejection Thresholds.

BACKGROUND: Using middleware solutions, it is possible to implement concentration-dependent analyte-specific hemolysis rejection limits. This makes day-to-day reporting of clinical specimens more efficient and potentially lowers sample rejection rates compared to a "one-size-fits-all" approach (i.e., solely based on a single cutoff provided in the package insert). METHODS: Hemolysis interference studies were performed at multiple analyte concentrations for three frequently ordered tests. For each assay, concentration-dependent hemolysis rejection limits were designed based on the total allowable error (TAE) for the analyte as well as the clinical significance of such incurred inaccuracy at the respective concentrations. In general, the following rationale was used: if the interference exceeds 10% (or package insert cutoffs), a comment is placed on the result. If the interference exceeds the TAE, the result will not be reported. Reduction in specimen rejection rates were estimated by comparing the incurred specimen rejection rates when package inserts' vs concentration-dependent hemolysis interference limits were applied to a data set in our institute during a three-month period. RESULTS: Concentration-dependent analyte-specific hemolysis rejection thresholds were designed for three commonly ordered assays that are especially susceptible to hemolysis interference. It is estimated that these novel thresholds for aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and direct bilirubin (DBIL) reduced specimen rejection rates from 9.3% to 1.3%, 31.4% to 4.8%, and 19.9% to 7.1%, respectively. CONCLUSIONS: Concentration-dependent analyte-specific hemolysis rejection thresholds for three commonly ordered assays can reduce rejection rates without significantly compromising the quality of test results.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.

A suspected case of falsely low digoxin and vancomycin concentrations caused by free kappa light chains with PETINIA method.

Vancomycin and digoxin are associated with potential toxicity and serum concentrations need to be monitored in certain patients. Previous reports suggested IgM paraproteins could interfere with vancomycin assays, and no paraprotein interference has been reported with digoxin assays. Here we present a suspected case of free-kappa light chains-mediated falsely low digoxin and vancomycin concentrations with Abbott particle-enhanced turbidimetric inhibition immunoassay (PETINIA) method. A 53-year-old patient received multiple doses of vancomycin and digoxin intravenously, but trough vancomycin and random digoxin concentrations repeatedly measured as <1.1 µg/mL and <0.2 ng/mL respectively with Abbott PETINIA method. Results from alternative methods showed concentrations reaching toxic levels and administration of the drugs was immediately terminated. A significantly elevated level of free-kappa light chains, possibly in polymeric form as suggested by protein electrophoresis result, was suspected to be the cause of falsely low results. During the laboratory investigation, absorbance curves revealed increased agglutination in the patient's samples in the latter part of the reaction, suggesting interfering substances led to production of turbidity after reagents were added. Protein-free filtration partially recovered the drugs with Abbott PETINIA. When drug concentrations do not correlate with clinical judgment, clinicians and pharmacists should consult clinical laboratories for investigation of potential interfering substances.

Validation of a Large Custom-Designed Pharmacogenomics Panel on an Array Genotyping Platform.

BACKGROUND: Pharmacogenomics has the potential to improve patient outcomes through predicting drug response. We designed and evaluated the analytical performance of a custom OpenArray® pharmacogenomics panel targeting 478 single-nucleotide variants (SNVs). METHODS: Forty Coriell Institute cell line (CCL) DNA samples and DNA isolated from 28 whole-blood samples were used for accuracy evaluation. Genotyping calls were compared to at least 1 reference method: next-generation sequencing, Sequenom MassARRAY®, or Sanger sequencing. For precision evaluation, 23 CCL samples were analyzed 3 times and reproducibility of the assays was assessed. For sensitivity evaluation, 6 CCL samples and 5 whole-blood DNA samples were analyzed at DNA concentrations of 10 ng/µL and 50 ng/µL, and their reproducibility and genotyping call rates were compared. RESULTS: For 443 variants, all samples assayed had concordant calls with at least 1 reference genotype and also demonstrated reproducibility. However, 6 of these 443 variants showed an unsatisfactory performance, such as low PCR amplification or insufficient separation of genotypes in scatter plots. Call rates were comparable between 50 ng/µL DNA (99.6%) and 10 ng/µL (99.2%). Use of 10 ng/µL DNA resulted in an incorrect call for a single sample for a single variant. Thus, as recommended by the manufacturer, 50 ng/µL is the preferred concentration for patient genotyping. CONCLUSIONS: We evaluated a custom-designed pharmacogenomics panel and found that it reliably interrogated 437 variants. Clinically actionable results from selected variants on this panel are currently used in clinical studies employing pharmacogenomics for clinical decision-making.

Use of the angiogenic biomarker profile to risk stratify patients with fetal growth restriction.

BACKGROUND: Novel angiogenic biomarker profiles have demonstrated emerging evidence for predicting preeclampsia onset, severity, and adverse outcomes. Limited data exist in screening patients with fetal growth restriction for preeclampsia development using angiogenic biomarkers. OBJECTIVE: The objective of this study was to risk stratify patients with fetal growth restriction using a soluble fms-like tyrosine kinase-1 to placental growth factor ratio. Previously published cutoff of 38 was used to predict preeclampsia development and severity as well as adverse maternal or neonatal outcomes within a 2-week time period. STUDY DESIGN: This was a prospective observational cohort study performed in a single tertiary hospital. Patients with a singleton fetal growth restriction pregnancy between 24 and 37 weeks' gestation were evaluated using serial 2-week encounters from the time of enrollment to delivery. Pregnancies with proven genetic or infectious etiology of fetal growth restriction or congenital anomalies were excluded. Ultrasound growth and Doppler measurements were obtained at the start of every encounter with routine preeclampsia laboratory tests and blood pressure checks when clinically indicated. Maternal serum was collected for all serial encounters and measured for soluble fms-like tyrosine kinase-1 and placental growth factor after delivery in a double-blinded fashion. Maternal charts were reviewed for baseline demographic characteristics, pregnancy diagnoses and outcomes, and neonatal outcomes. RESULTS: A total of 45 patients were enrolled for a total of 77 encounters, with the median (quartile 1, quartile 3) gestational age of the study enrolled at 31.43 (28.14-33.57) weeks. Patients were divided into low-risk (ratio of <38) and high-risk (ratio of ≥38) groups. Baseline characteristics of patients did not show any marked differences, including preeclampsia labs or ultrasound parameters, between the 2 groups. Systolic and diastolic blood pressures upon enrollment were statistically elevated when soluble fms-like tyrosine kinase-1 to placental growth factor ratio was ≥38 (P=.02 and P=.01, respectively). Compared to patients with a low ratio, patients with a high ratio had a greater proportion of preeclampsia diagnosis, higher rates of preterm delivery under 34 and 37 weeks gestation, smaller neonatal birthweight, and a shorter time to delivery from testing to delivery. CONCLUSION: Among patients with fetal growth restriction, the soluble fms-like tyrosine kinase-1 to placental growth factor ratio may serve as a potential biomarker for identifying at risk patients for developing preeclampsia and subsequently preterm delivery.

Analytical and Clinical Evaluation of the Automated Elecsys Anti-SARS-CoV-2 Antibody Assay on the Roche cobas e602 Analyzer.

OBJECTIVES: To evaluate the analytical and clinical performance of the automated Elecsys anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody (Elecsys Ab) assay on the Roche cobas e602 analyzer. With the ongoing global coronavirus disease 2019 (COVID-19) pandemic, widespread and routine serologic testing of SARS-CoV-2 remains a pressing need. To better understand its epidemiologic spread and to support policies aimed at curtailing further infections, reliable serologic testing is crucial for providing insight into the dynamics of the spread of COVID-19 on a population level. METHODS: The presence of anti-SARS-CoV-2 antibodies in polymerase chain reaction-positive, confirmed COVID-19 patient samples was determined using the Elecsys Ab assay on the Roche cobas e602 analyzer. The precision and cross-reactivity of the Elecsys Ab assay were characterized and its performance was compared against the EuroImmun IgA/IgG antibody (EuroImmun Ab) assay. Calculated sensitivity, specificity, and positive and negative predictive values were assessed. RESULTS: The Elecsys Ab assay demonstrated good precision, had no cross-reactivity with other viral samples, and showed 100% concordance with the EuroImmun Ab assay. Excellent clinical performance with respect to sensitivity, specificity, and positive and negative predictive values was observed. CONCLUSIONS: The Elecsys Ab assay is a precise and highly reliable automated platform for clinical detection of seropositivity in SARS-CoV-2 infection.

A comparison of venous blood gas analysis with paired specimens collected in syringes and evacuated blood collection tubes.

BACKGROUND: Venous blood samples collected in evacuated blood collection tubes are generally not considered suitable for measurement of oxygenation status. However, whether pH, pCO2 and HCO3- results from venous blood samples collected in evacuated tubes are reliable remains unclear. METHODS: Paired samples were collected from 38 healthy volunteers using two collection methods: (1) collection of blood directly into a blood gas syringe and (2) collection of blood into an evacuated tube with subsequent anaerobic transfer into a blood gas syringe. Samples were analyzed for pH, pCO2, HCO3-, and pO2. RESULTS: Samples collected in evacuated tubes showed significant positive mean biases of 0.03 and 7.5 mmHg for pH and pO2, respectively, and significant negative mean biases of 5.0 mmHg and 1.2 mmol/l for pCO2 and HCO3-, respectively. Collection in evacuated tubes resulted in biases exceeding the total allowable errors in 16%, 40%, 21% of samples for pH, pCO2, HCO3-, respectively, when compared to samples collected in syringes directly. CONCLUSIONS: Samples for venous blood gas analysis should be collected directly into blood gas syringes for accurate assessment of ventilation and/or acid-base status. Biases observed for samples collected in evacuated tubes are consistent with air contamination and/or vacuum effects.

Fatty-Acid Uptake in Prostate Cancer Cells Using Dynamic Microfluidic Raman Technology.

It is known that intake of dietary fatty acid (FA) is strongly correlated with prostate cancer progression but is highly dependent on the type of FAs. High levels of palmitic acid (PA) or arachidonic acid (AA) can stimulate the progression of cancer. In this study, a unique experimental set-up consisting of a Raman microscope, coupled with a commercial shear-flow microfluidic system is used to monitor fatty acid uptake by prostate cancer (PC-3) cells in real-time at the single cell level. Uptake of deuterated PA, deuterated AA, and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were monitored using this new system, while complementary flow cytometry experiments using Nile red staining, were also conducted for the validation of the cellular lipid uptake. Using this novel experimental system, we show that DHA and EPA have inhibitory effects on the uptake of PA and AA by PC-3 cells.

The Ps and Qs of alarmone synthesis in Staphylococcus aureus.

During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically modulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, ß = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio.

Antibiotic susceptibility and molecular epidemiology of Acinetobacter calcoaceticus-baumannii complex strains isolated from a referral hospital in northern Vietnam.

Acinetobacter calcoaceticus-baumannii complex is a common cause of hospital-acquired infections (HAIs) globally, remarkable for its high rate of antibiotic resistance, including to carbapenems. There are few data on the resistance of A. baumannii in Vietnam, which are essential for developing evidence-based treatment guidelines for HAIs. Antibiotic susceptibility testing was conducted by VITEK®2, and pulsed-field gel electrophoresis (PFGE) was performed on 66 clinical A. baumannii complex isolates recovered during 2009 at the National Hospital of Tropical Diseases (NHTD), a referral hospital in Hanoi, Vietnam. Basic demographic and clinical data were collected and analysed using descriptive statistics. Most isolates came from lower respiratory tract specimens (59; 89.4%) from intensive care unit (ICU) patients [64/65 (98.5%) with available data] who had been admitted to NHTD for ≥2 days [42/46 (91.3%) with available data]. More than 90% of the isolates were resistant to the tested ß-lactamase/ß-lactamase inhibitors, cephalosporins, carbapenems, fluoroquinolones and trimethoprim/sulfamethoxazole. Moreover, 25.4% (16/63) were resistant to all tested ß-lactams, quinolones and aminoglycosides. All isolates remained sensitive to colistin and 58.7% were susceptible to tigecycline. Of the 66 isolates, 49 could be classified into eight PFGE types (A-H). Every PFGE type, except D, had cluster(s) of three or more isolates with a temporal relationship. In conclusion, these data suggest a significant rise in A. baumannii antibiotic resistance in Vietnam. Clustering within PFGE types supports cross-transmission of A. baumannii within the ICU at NHTD. Increased research and resources in optimising treatment, infection control and antibiotic stewardship are needed.