Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell ...repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.
In an extension of the pivotal randomized trial showing vaccine efficacy, approximately 10,000 adults who had received two doses of BNT162b2 were assigned to receive a third dose or placebo. Local ...reactions were common but mild and transient. During a median follow-up of 2.5 months, SARS-CoV-2 was identified in 6 participants in the vaccine group and in 123 in the placebo group, for a vaccine efficacy of 95.3%.
Generalized periodic discharges are a repeated and generalized electroencephalography (EEG) pattern that can be seen in the context of altered mental status. This article describes a series of five ...individuals with generalized periodic discharges who demonstrated signs and symptoms of catatonia, a treatable neuropsychiatric condition.
Inpatients with a clinical diagnosis of catatonia, determined with the Bush-Francis Catatonia Rating Scale (BFCRS), and EEG recordings with generalized periodic discharges were analyzed in a retrospective case series.
Five patients with catatonia and generalized periodic discharges on EEG were evaluated from among 106 patients with catatonia and contemporaneous EEG measurements. Four of these patients showed an improvement in catatonia severity when treated with benzodiazepines, with an average reduction of 6.75 points on the BFCRS.
Among patients with generalized periodic discharges, catatonia should be considered, in the appropriate clinical context. Patients with generalized periodic discharges and catatonia may benefit from treatment with empiric trials of benzodiazepines.
Particle image velocimetry is a powerful and flexible fluid velocity measurement tool. In spite of its widespread use, the uncertainty of PIV measurements has not been sufficiently addressed to date. ...The calculation and propagation of local, instantaneous uncertainties on PIV results into the measured mean and Reynolds stresses are demonstrated for four PIV error sources that impact uncertainty through the vector computation: particle image density, diameter, displacement and velocity gradients. For the purpose of this demonstration, velocity data are acquired in a rectangular jet. Hot-wire measurements are compared to PIV measurements with velocity fields computed using two PIV algorithms. Local uncertainty on the velocity mean and Reynolds stress for these algorithms are automatically estimated using a previously published method. Previous work has shown that PIV measurements can become 'noisy' in regions of high shear as well as regions of small displacement. This paper also demonstrates the impact of these effects by comparing PIV data to data acquired using hot-wire anemometry, which does not suffer from the same issues. It is confirmed that flow gradients, large particle images and insufficient particle image displacements can result in elevated measurements of turbulence levels. The uncertainty surface method accurately estimates the difference between hot-wire and PIV measurements for most cases. The uncertainty based on each algorithm is found to be unique, motivating the use of algorithm-specific uncertainty estimates.
Optimizing pulmonary health across the lifespan begins from the earliest stages of childhood and requires a partnership between the family, pulmonologist, and pediatrician to achieve equitable ...outcomes. The Community Pediatrics session of the Defining and Promoting Pediatric Pulmonary Health workshop weaved together 4 community-based pillars with 4 research principles to set an agenda for future pediatric pulmonary research in optimizing lung and sleep health for children and adolescents. To address diversity, equity, and inclusion, both research proposals and workforce must purposefully include a diverse set of participants that reflects the community served, in addition to embracing nontraditional, community-based sites of care and social determinants of health. To foster inclusive, exploratory, and innovative research, studies must be centered on community priorities, with findings applied to all members of the community, particularly those in historically marginalized and minoritized groups. Research teams should also foster meaningful partnerships with community primary care and family members from study conceptualization. To achieve these goals, implementation and dissemination science should be expanded in pediatric pulmonary research, along with the development of rapid mechanisms to disseminate best practices to community-based clinicians. To build cross-disciplinary collaboration and training, community–academic partnerships, family research partnerships, and integrated research networks are necessary. With research supported by community pillars built on authentic partnerships and guided by inclusive principles, pediatric lung and sleep health can be optimized for all children and adolescents across the full lifespan in the community in which they live and thrive.
We investigated expression of genes mediating elemental cycling at the microspatial scale in the ocean’s largest river plume using, to our knowledge, the first fully quantitative inventory of genes ...and transcripts. The bacterial and archaeal communities associated with a phytoplankton bloom in Amazon River Plume waters at the outer continental shelf in June 2010 harbored ∼1.0 × 10 ¹³ genes and 4.7 × 10 ¹¹ transcripts per liter that mapped to several thousand microbial genomes. Genomes from free-living cells were more abundant than those from particle-associated cells, and they generated more transcripts per liter for carbon fixation, heterotrophy, nitrogen and phosphorus uptake, and iron acquisition, although they had lower expression ratios (transcripts⋅gene ⁻¹) overall. Genomes from particle-associated cells contributed more transcripts for sulfur cycling, aromatic compound degradation, and the synthesis of biologically essential vitamins, with an overall twofold up-regulation of expression compared with free-living cells. Quantitatively, gene regulation differences were more important than genome abundance differences in explaining why microenvironment transcriptomes differed. Taxa contributing genomes to both free-living and particle-associated communities had up to 65% of their expressed genes regulated differently between the two, quantifying the extent of transcriptional plasticity in marine microbes in situ. In response to patchiness in carbon, nutrients, and light at the micrometer scale, Amazon Plume microbes regulated the expression of genes relevant to biogeochemical processes at the ecosystem scale.
A surface plasmon polariton (SPP) is an excitation resulting from the coupling of light to a surface charge oscillation at a metal-dielectric interface. The excitation and detection of SPPs is ...foundational to the operating mechanism of a number of important technologies, most of which require SPP excitation via direct reflectance, commonly achieved via Attenuated Total Reflection (ATR) using the Kretschmann configuration. As a result, the accessible modes are fundamentally high-loss “leaky modes,” presenting a critical performance barrier. Recently, our group provided the first demonstration of “forbidden,” or guided-wave plasmon polariton modes (GW-PPMs), collective modes of a MIM structure with oscillatory electric field amplitude in the central insulator layer with up to an order of magnitude larger propagation lengths than those of traditional SPPs. However, in that work, GW-PPMs were accessed by indirect reflectance using Otto configuration ATR, making them of limited applied relevance. In this paper, we demonstrate a technique for direct reflectance excitation and detection of GW-PPMs. Specifically, we replace the air gap used in traditional Otto ATR with a low refractive index polymer coupling layer, mirroring a technique previously demonstrated to access Long-Range Surface Plasmon Polariton modes. We fit experimental ATR data using a robust theoretical model to confirm the character of the modes, as well as to explore the potential of this approach to enable advantageous propagation lengths. The ability to excite GW-PPMs using a device configuration that does not require an air gap could potentially enable transformative performance enhancements in a number of critical technologies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, ...time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by
. In fact, in a Δ
strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the Δ
strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment.
Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism's phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.
Following changes in vegetation structure and pattern, along with a changing climate, large wildfire incidence has increased in forests throughout the western United States. Given this increase, ...there is great interest in whether fuels treatments and previous wildfire can alter fire severity patterns in large wildfires. We assessed the relative influence of previous fuels treatments (including wildfire), fire weather, vegetation, and water balance on fire-severity in the Rim Fire of 2013. We did this at three different spatial scales to investigate whether the influences on fire severity changed across scales. Both fuels treatments and previous low to moderate-severity wildfire reduced the prevalence of high-severity fire. In general, areas without recent fuels treatments and areas that previously burned at high severity tended to have a greater proportion of high-severity fire in the Rim Fire. Areas treated with prescribed fire, especially when combined with thinning, had the lowest proportions of high severity. The proportion of the landscape burned at high severity was most strongly influenced by fire weather and proportional area previously treated for fuels or burned by low to moderate severity wildfire. The proportion treated needed to effectively reduce the amount of high severity fire varied by spatial scale of analysis, with smaller spatial scales requiring a greater proportion treated to see an effect on fire severity. When moderate and high-severity fire encountered a previously treated area, fire severity was significantly reduced in the treated area relative to the adjacent untreated area. Our results show that fuels treatments and low to moderate-severity wildfire can reduce fire severity in a subsequent wildfire, even when burning under fire growth conditions. These results serve as further evidence that both fuels treatments and lower severity wildfire can increase forest resilience.
Introduction
Bariatric surgery improves many obesity-related comorbidities, yet the literature remains inconclusive on the impact of bariatric surgery on asthma. Our primary objective was to identify ...the long-term impact of bariatric surgery on asthma severity and medication use.
Methods
A retrospective review was completed of all patients with a diagnosis of asthma who underwent bariatric surgery over 10 years at a single institution. Primary outcomes were the number of asthma medications prescribed at five time points (preoperative, postoperative < 18 months, 19–36 months, 37–60 months, 60 + months) after bariatric surgery. Secondary outcomes were spirometry results and BMI.
Results
There were 260 patients with 84.6% female predominance. There were 168 sleeve gastrectomy patients and 92 Roux-en-Y gastric bypass patients. Mean age was 47.6 ± 10.7 years, mean BMI was 46.0 ± 6.8 kg/m
2
, and 54.2% were previous tobacco users. The total number of patients on two or more asthma medications decreased from 46% preoperatively to 41% at 18 months, to 36% at 36 months, and to 32% at 60 months after surgery. The total number of patients free from asthma medication increased from 25% preoperatively to 33% at 60 months postoperatively. Asthma medication use decreased in both surgery groups, and neither operation demonstrated superiority. No significant improvement nor differences were found between groups at any time point regarding FEV
1
/FVC ratio spirometry measures.
Conclusion
Bariatric surgery reduces the use of medications taken for management of asthma. The amount of asthma medication usage decreases with time and is sustained at 60 months after bariatric surgery.
Graphical Abstract
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