Development and validation of methods that enable high-quality droplet digital PCR and hematological profiling data from microvolume blood samples.

Aim: Mouse models have been crucial to preclinical studies in the increasingly relevant fields of cell and gene therapy. However, only small quantities of mouse blood can be collected without producing adverse physiological effects that compromise data integrity. Results: To address this limitation, two combined methods were developed to create detailed droplet digital PCR (ddPCR) and hematological profiles using only ∼20 µl of mouse blood. The validation of these methods, which can serve as a foundation for a standardized regulatory pipeline for ddPCR, is discussed. Even when using small amounts of input, this ddPCR protocol is accurate, precise, selective, specific, stable and robust. Conclusion: These techniques enable more frequent sample collection for higher-resolution pharmacokinetic data that meets or exceeds quality standards.

This article addresses new experimental methods that optimize two distinct techniques. The first technique, known as droplet digital PCR (ddPCR), is a process where DNA is isolated in oil droplets, cloned and then counted. The second technique is known as hematological profiling, in which various components of blood are counted (e.g., red blood cells, hemoglobin). Although ddPCR is incredibly promising as a research tool, there are a few hurdles that limit its usage. First, as of the time of writing, regulatory bodies (i.e., FDA and EMA) have not published official or standardized guidelines specific for either ddPCR or a closely related technique known quantitative PCR (qPCR). This has been an impetus for scientists to independently adopt and abide by unofficial recommendations for ddPCR and to negotiate with these regulatory bodies without a mutually accepted reference point between the two parties. Second, ddPCR can consume considerable volumes of blood, which can impair overall health; this limits how often samples can be taken, making scientists less effective at tracking how much medicine remains in the bloodstream over time. The new methods in this article use less blood, addressing these concerns and allowing scientists to safely collect blood from subjects more often, while also meeting all regulatory criteria established for similar scientific techniques and following informal and widely agreed upon scientific recommendations. Therefore, this work can serve as an easily adaptable framework for most ddPCR experiments.

The small GTPase RAB-35 facilitates the initiation of phagosome maturation and acts as a robustness factor for apoptotic cell clearance.

We recently identified the novel function of the small GTPase RAB-35 in apoptotic cell clearance in Caenorhabditis elegans, a process in which dying cells are engulfed and degraded inside phagosomes. We have found that RAB-35 functions in two separate steps of cell corpse clearance, cell corpse recognition and the initiation of phagosome maturation. During the latter process, RAB-35 facilitates the removal of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the membranes of nascent phagosomes and the simultaneous production of phosphatidylinositol-3-P (PI(3)P) on these same membranes, a process that we have coined the PI(4,5)P2 to PI(3)P shift. RAB-35 also promotes the recruitment of the small GTPase RAB-5 to the phagosomal surface. During these processes, the activity of RAB-35 is controlled by the candidate GTPase-activating protein (GAP) TBC-10 and the candidate guanine nucleotide exchange factor (GEF) FLCN-1. Overall, RAB-35 leads a third pathway during cell corpse clearance that functions in parallel to the two known pathways, one led by the phagocytic receptor CED-1 and the other led by the CED-10/Rac1 GTPase. Here, we further report that RAB-35 acts as a robustness factor that maintains the clearance activity and embryonic viability under conditions of heat stress. Moreover, we obtained additional evidence suggesting that RAB-35 acts upstream of RAB-5 and RAB-7. To establish a precise temporal pattern for its own dissociation from phagosomal surfaces, RAB-35 controls the removal of its own GAP. We propose that RAB-35 defines a largely unexplored initial phase of phagosome maturation.

Chronic Obstructive Pulmonary Disease in the Intensive Care Unit: Antibiotic Treatment of Severe Chronic Obstructive Pulmonary Disease Exacerbations.

Patients who suffer from chronic obstructive pulmonary disease (COPD) often experience deterioration of baseline respiratory symptoms, acute exacerbations of COPD (AECOPD), that become more frequent with disease progression. Based on symptom severity, approximately 20% of these patients will require hospitalization. The most common indicators for intensive care unit (ICU) admission have been found to be worsening or impending respiratory failure and hemodynamic instability. Bacterial and viral bronchial infections are the causative triggers in the majority of COPD exacerbations in the ICU, with a comprehensive assessment revealing them in 72% of cases. The distribution of bacterial pathogens involved in AECOPD requiring ICU admission show an increased incidence of gram-negative respiratory isolates, including Pseudomonas and Enterobacteriaceae spp., when compared with outpatient exacerbations. Evaluation of these patients requires careful attention to comorbid conditions. An effort to rapidly obtain lower respiratory samples for microbiological samples prior to initiation of antibiotics should be made as adequate samples can guide subsequent modifications of antibiotic treatment if the clinical response to empiric treatment is poor. Empiric antibiotic treatment should be promptly initiated in all patients with a major consideration for the choice being the presence of risk factors for Pseudomonas infection. Evaluation of clinical response at 48 to 72 hours is crucial, and total duration of antibiotics of 5 to 7 days should be adequate.

Aclidinium bromide and formoterol fumarate for the maintenance treatment of chronic obstructive pulmonary disease.

Introduction: Treatment options for COPD have evolved rapidly in the last decade and inhaled bronchodilators have largely supplanted the use of oral bronchodilators because of their increased efficacy and excellent safety with topical delivery to the lung. Recently added to the therapeutic armamentarium are fixed-dose combinations (FDC) of two long acting bronchodilators. LAMAs (long acting muscarinic antagonists) and LABAs (long acting beta agonists) are the main classes available and use different pathways to effectively produce bronchial smooth muscle relaxation.Areas covered: The most recent inhaled FDC LAMA/LABA to come to market is Aclidinium Bromide and Formoterol Fumarate. We searched databases of PubMed, Cochrane Library, and manufacturers' websites and retrieved all the randomized-controlled trials (RCTs) conducted with these drugs up to September 2019.Expert opinion: It is likely that FDCs will become the core of our COPD pharmacotherapy for all but the mildest COPD patients. These individual drugs have excellent efficacy and safety records for the maintenance treatment of COPD. Studies have demonstrated that twice daily treatment with aclidinium/formoterol resulted in significant improvement in lung function and an improved exercise tolerance when compared to placebo. Adverse effects are within the range of what is seen with other LAMA/LABA combinations.

The small GTPase RAB-35 defines a third pathway that is required for the recognition and degradation of apoptotic cells.

In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells.

Myocardial computed tomography perfusion.

Despite having excellent diagnostic accuracy to detect anatomical coronary stenosis, coronary CT angiography (CTA) has a limited specificity to detect myocardial ischemia. CT perfusion (CTP) can identify myocardial perfusion defects during vasodilator stress, and when added to coronary CTA, improves the specificity of detecting hemodynamically significant stenosis. A CTP protocol typically involves the acquisition of two separate data sets: (I) a rest scan that can be used as both a coronary CTA and for evaluating rest myocardial perfusion, and (II) a stress CTP scan acquired during vasodilator stress testing. This review summarizes some the techniques, strengths, and limitations of CTP, and provides an overview of the recent evidence supporting the potential use of CTP in clinical practice.

Cardiovascular Imaging for the Primary Prevention of Atherosclerotic Cardiovascular Disease Events.

Traditional cardiovascular risk factors have well-known limitations for the accurate assessment of individual cardiovascular risk. Unlike risk factor-based scores which rely on probabilistic calculations derived from population-based studies, coronary artery calcium (CAC) scoring, and carotid ultrasound allow for the direct visualization and quantification of subclinical atherosclerosis with the potential for a more accurate, personalized risk assessment and treatment approach. Among strategies used to guide preventive management, CAC scoring has consistently and convincingly outperformed traditional risk factors for the prediction of adverse cardiovascular events. Moreover, several studies have demonstrated the potential of CAC testing to improve precision for the use of more intensive pharmacologic therapies, such as aspirin and statins, in patients most likely to derive benefit, as compared to atherosclerotic cardiovascular disease risk calculators. By comparison to CAC, the role of carotid ultrasound for the measurement of carotid intima-media thickness (CIMT) remains less well-elucidated but may be significantly improved with the inclusion of plaque screening and novel three-dimensional measurements of plaque volume and morphology. Despite significant evidence supporting the ability of non-invasive atherosclerosis imaging (particularly CAC) to guide preventive management, imaging remains an under-utilized strategy among current guidelines and clinical practice. Herein, we review evidence regarding CAC and carotid ultrasound for patient risk classification, with a comparison of these techniques to currently advocated traditional risk factor-based scores.

Identification and characterization of native proteins of Escherichia coli BL-21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices.

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E. coli proteins. Nine out of the ten have isoelectric values less than six, and half are considered nonessential. These data indicate that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC-HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome.

Dynamics of malaria drug resistance patterns in the Amazon basin region following changes in Peruvian national treatment policy for uncomplicated malaria.

Monitoring changes in the frequencies of drug-resistant and -sensitive genotypes can facilitate in vivo clinical trials to assess the efficacy of drugs before complete failure occurs. Peru changed its national treatment policy for uncomplicated malaria to artesunate (ART)-plus-mefloquine (MQ) combination therapy in the Amazon basin in 2001. We genotyped isolates collected in 1999 and isolates collected in 2006 to 2007 for mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, multidrug resistance gene 1 (Pfmdr-1), the chloroquine (CQ) resistance transporter gene (Pfcrt), and the Ca(2+) ATPase gene (PfATP6); these have been shown to be involved in resistance to sulfadoxine-pyrimethamine (SP), MQ, CQ, and possibly ART, respectively. Microsatellite haplotypes around the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr-1 loci were also determined. There was a significant decline in the highly SP resistant Pfdhfr and Pfdhps genotypes from 1999 to 2006. In contrast, a CQ-resistant Pfcrt genotype increased in frequency during the same period. Among five different Pfmdr-1 allelic forms noted in 1999, two genotypes increased in frequency while one genotype decreased by 2006. We also noted previously undescribed polymorphisms in the PfATP6 gene as well as an increase in the frequency of a deletion mutant during this period. In addition, microsatellite analysis revealed that the resistant Pfdhfr, Pfdhps, and Pfcrt genotypes have each evolved from a single founder haplotype, while Pfmdr-1 genotypes have evolved from at least two independent haplotypes. Importantly, this study demonstrates that the Peruvian triple mutant Pfdhps genotypes are very similar to those found in other parts of South America.