Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

Aptamer-graphene oxide for highly sensitive dual electrochemical detection of Plasmodium lactate dehydrogenase

A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by I_D/I_G intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

Determination of community structure through deconvolution of PLFA-FAME signature of mixed population

Phospholipid fatty acids (PLFAs) as biomarkers are well established in the literature. A general method based on least square approximation (LSA) was developed for the estimation of community structure from the PLFA signature of a mixed population where biomarker PLFA signatures of the component species were known. Fatty acid methyl ester (FAME) standards were used as species analogs and mixture of the standards as representative of the mixed population. The PLFA/FAME signatures were analyzed by gas chromatographic separation, followed by detection in flame ionization detector (GC-FID). The PLFAs in the signature were quantified as relative weight percent of the total PLFA. The PLFA signatures were analyzed by the models to predict community structure of the mixture. The LSA model results were compared with the existing "functional group" approach. Both successfully predicted community structure of mixed population containing completely unrelated species with uncommon PLFAs. For slightest intersection in PLFA signatures of component species, the LSA model produced better results. This was mainly due to inability of the "functional group" approach to distinguish the relative amounts of the common PLFA coming from more than one species. The performance of the LSA model was influenced by errors in the chromatographic analyses. Suppression (or enhancement) of a component's PLFA signature in chromatographic analysis of the mixture, led to underestimation (or overestimation) of the component's proportion in the mixture by the model. In mixtures of closely related species with common PLFAs, the errors in the common components were adjusted across the species by the model.     

Cyclin-dependent kinase inhibitors sensitize tumor cells to nutlin-induced apoptosis: a potent drug combination

Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose- and time-dependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser(15) on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage-mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy.     

Evolution of chromosome number in grasshoppers (Orthoptera: Caelifera: Acrididae)

Organisms Diversity & Evolution - Orthoptera have some of the largest genomes of all insects. At the same time, the architecture of their genomes remains poorly understood. Comparative...     

Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with V_max of 3.9 ± 0.2 μmol/min/mg and K _m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN–PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN–PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN–PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN–PscL complex in T3SS mediated pathogenic pathway.     

Determining clinically relevant features in cytometry data using persistent homology

Cytometry experiments yield high-dimensional point cloud data that is difficult to interpret manually. Boolean gating techniques coupled with comparisons of relative abundances of cellular subsets is the current standard for cytometry data analysis. However, this approach is unable to capture more subtle topological features hidden in data, especially if those features are further masked by data transforms or significant batch effects or donor-to-donor variations in clinical data. We present that persistent homology, a mathematical structure that summarizes the topological features, can distinguish different sources of data, such as from groups of healthy donors or patients, effectively. Analysis of publicly available cytometry data describing non-naïve CD8+ T cells in COVID-19 patients and healthy controls shows that systematic structural differences exist between single cell protein expressions in COVID-19 patients and healthy controls. We identify proteins of interest by a decision-tree based classifier, sample points randomly and compute persistence diagrams from these sampled points. The resulting persistence diagrams identify regions in cytometry datasets of varying density and identify protruded structures such as ‘elbows’. We compute Wasserstein distances between these persistence diagrams for random pairs of healthy controls and COVID-19 patients and find that systematic structural differences exist between COVID-19 patients and healthy controls in the expression data for T-bet, Eomes, and Ki-67. Further analysis shows that expression of T-bet and Eomes are significantly downregulated in COVID-19 patient non-naïve CD8+ T cells compared to healthy controls. This counter-intuitive finding may indicate that canonical effector CD8+ T cells are less prevalent in COVID-19 patients than healthy controls. This method is applicable to any cytometry dataset for discovering novel insights through topological data analysis which may be difficult to ascertain otherwise with a standard gating strategy or existing bioinformatic tools.