Defining the pathway of worm-like amyloid fibril formation by the mouse prion protein by delineation of the productive and unproductive oligomerization reactions

Aggregation reactions of proteins leading to amyloid fibril formation are often characterized by early transient accumulation of a heterogeneous population of soluble oligomers differing in size and structure. Delineating the kinetic roles of the different oligomeric forms in fibril formation has been a major challenge. The aggregation of the mouse prion protein to form worm-like amyloid fibrils at low pH is known to proceed via a β-rich oligomer ensemble, which is shown here to be comprised of two subpopulations of oligomers that differ in size and internal structure. The relative populations of the two oligomers can be tuned by varying the concentration of NaCl present. By demonstrating that the apparent rate constant for the formation of fibrils is dependent linearly on the concentration of the larger oligomer and is independent of the concentration of the smaller oligomer, we show that the larger oligomer is a productive intermediate that accumulates on the direct pathway of aggregation from monomer to worm-like fibrils. The smaller oligomer is shown to be populated off the pathway of the larger oligomer and, hence, is not directly productive for fibril formation. The relative populations of the two oligomers can also be tuned by single-amino acid residue changes in the sequence of the protein. The different protein variants yield worm-like fibrils of different lengths, and the apparent rate of formation of the fibrils by the mutant variants is also shown to be dependent on the concentration of the larger but not of the smaller oligomer formed.     

Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.     

Dissection of Conformational Conversion Events during Prion Amyloid Fibril Formation Using Hydrogen Exchange and Mass Spectrometry

A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH 7 has been characterized by hydrogen–deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen–deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~ 159–225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109–132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis.     

Establishment of Elevated Serum Levels of IL-10, IL-8 and TNF-β as Potential Peripheral Blood Biomarkers in Tubercular Lymphadenitis: A Prospective Observational Cohort Study

Background

Tubercular lymphadenitis (TL) is the most common form of extra-pulmonary tuberculosis (TB) consisting about 15–20% of all TB cases. The currently available diagnostic modalities for (TL), are invasive and involve a high index of suspicion, having limited accuracy. We hypothesized that TL would have a distinct cytokine signature that would distinguish it from pulmonary TB (PTB), peripheral tubercular lymphadenopathy (LNTB), healthy controls (HC), other lymphadenopathies (LAP) and cancerous LAP. To assess this twelve cytokines (Tumor Necrosis Factor (TNF)—α, Interferon (IFN) -γ, Interleukin (IL)-2, IL-12, IL-18, IL-1β, IL-10, IL-6, IL-4, IL-1Receptor antagonist (IL-1Ra), IL-8 and TNF-β, which have a role in pathogenesis of tuberculosis, were tested as potential peripheral blood biomarkers to aid the diagnosis of TL when routine investigations prove to be of limited value.

Methods and Findings

A prospective observational cohort study carried out during 2010–2013. This was a multi-center study with three participating hospitals in Delhi, India where through random sampling cohorts were established. The subjects were above 15 years of age, HIV-negative with no predisposing ailments to TB (n = 338). The discovery cohort (n = 218) had LNTB (n = 50), PTB (n = 84) and HC (n = 84). The independent validation cohort (n = 120) composed of patients with cancerous LAP (n = 35), other LAP (n = 20) as well as with independent PTB (n = 30), LNTB (n = 15) and HC (n = 20). Eight out of twelve cytokines achieved statistical relevance upon evaluation by pairwise and ROC analysis. Further, variable selection using random forest backward elimination revealed six serum biosignatures including IL-12, IL-4, IL-6, IL-10, IL-8 and TNF-β as optimal for classifying the LNTB status of an individual. For the sake of clinical applicability we further selected a three analyte panel (IL-8, IL-10 and TNF-β) which was subjected to multinomial modeling in the independent validation cohort which was randomised into training and test cohorts, achieving an overwhelming 95.9% overall classifying accuracy for correctly classifying LNTB cases with a minimal (7%) misclassification error rate in the test cohort.

Conclusions

In our study, a three analyte serum biosignatures and probability equations were established which can guide the physician in their clinical decision making and step wise management of LNTB patients. This set of biomarkers has the potential to be a valuable adjunct to the diagnosis of TL in cases where AFB positivity and granulomatous findings elude the clinician.     

Production of Lactic Acid from Cellobiose and Cellotriose by Lactobacillus delbrueckii Mutant Uc-3

Lactobacillus delbrueckii mutant Uc-3 utilizes both cellobiose and cellotriose efficiently, converting it into L(+) lactic acid. The enzyme activities of cellobiose and cellotriose utilization were determined for cell extracts, whole cells, and disrupted cells. Aryl-β-glucosidase activity was detected only for whole cells and disrupted cells, suggesting that these activities are cell bound. The mutant produced 90 g/liter of lactic acid from 100 g/liter of cellobiose with 2.25 g/liter/h productivity.     

Guanidine hydrochloride-induced reversible unfolding of sheep liver serine hydroxymethyltransferase

Equilibrium unfolding studies of sheep liver tetrameric serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) revealed that the enzyme assumed apparent random coil structure above 3 M guanidine hydrochloride (GdnHCl). In the presence of non-ionic detergent Brij-35 and polyethylene glycol, the 6 M GdnHCI unfolded enzyme could be completely (> 95%) refolded by a 40-fold dilution. The refolded enzyme was fully active and had kinetic constants similar to the native enzyme. The midpoint of inactivation (0.12 M GdnHCl) was well below the midpoint of unfolding (1.6±0.1 M GdnHCl) as monitored by far UV CD at 222 nm. In the presence of PLP, the midpoint of inactivation shifted to a higher concentration of GdnHCl (0.6 M) showing that PLP stabilizes the quaternary structure of the enzyme. However, 50% release of pyridoxal-5′-phosphate (PLP) from the active site occurred at a concentration (0.6 M) higher than the midpoint of inactivation suggesting that GdnHCl may also act as a competitive inhibitor of the enzyme at low concentrations which was confirmed by activity measurements. PLP was not required for the initiation of refolding and inactive tetramers were the end products of refolding which could be converted to active tetramers upon the addition of PLP. Size exclusion chromatography of the apoenzyme showed that the tetramer unfolds via the intermediate formation of dimers. Low concentrations (0.3–0.6 M) of GdnHCl stabilized at least one intermediate which was in slow equilibrium with the dimer. The binding of ANS was maximum at 0.4–0.6 M GdnHCl suggesting that the unfolding intermediate that accumulates at this concentration is less compact than the native enzyme.     

Natural killer and antibody-dependent cellular cytotoxicity in cervical carcinoma patients

Summary Natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC) was measured in 62 untreated cervical carcinoma patients and 25 normal healthy women, using a short-term chromium release assay. A significant reduction in NK and ADCC activity was observed in disseminated disease than in localized disease, when compared with normal donors. The majority of the patients received radiotherapy and both NK and ADCC activity recovered after therapy. Furthermore, interferon- was demonstrated to augment NK activity of peripheral blood mononuclear cells from healthy donors as well as patients. Also large granular lymphocytes separated on Percoll density gradient were the same in number in both the populations studied, although in cervical cancer there seemed to be a defect in killing activity.