On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

<div><p>Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (<i>k</i> = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.</p></div

Activity and size of HEWL aggregates.

<p><b>a,</b> Bacteriolytic activity (in ▵Abs/min) of HEWL (diluted to 70 nM in assay cuvette) measured at pH 7 with pppppppwith <i>M. lysodeikticus</i> is highlighted at different time intervals subsequent to incubation of HEWL at concentrations 0.7, 3, 50 & 120 µM in pH 12.2 and 120 µM in pH 7. <b>b</b>, Changes in steady state fluorescence anisotropy (r_ss) of dansyl-HEWL conjugates at different time intervals in presence of 0.3, 3, 20, 50 & 120 µM of HEWL incubated in pH 12.2 and 50 µM in pH 7 is shown along with fitted curve for pH 12.2 (Eq. 2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087256#pone-0087256-t001" target="_blank">Table 1</a>). <b>c</b>, Nanosecond time-resolved fluorescence anisotropy decay of dansyl-HEWL conjugates after 24 hours for following protein concentrations (pH): A: 3 µM (7); B—E: 0.3, 3, 20 and 120 µM (12.2). The dark continuous line depicts the fit and extracted value for φ₂ using tail fit analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087256#pone.0087256.s007" target="_blank">Table S1</a>). <b>d</b>, Average global rotational time (φ₂) for 0.3—120 µM HEWL at pH 12.2 and 3 µM at pH 7 (extreme left) after 12 & 24 hours (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087256#pone.0087256.s008" target="_blank">Table S2</a>). <b>e</b>, Size of HEWL aggregates revealed by dynamic light scattering after incubation of 120 µM HEWL in pH 12.2 (continuous line) and pH 7 (dashed line) for 26 hours. Peaks at R_h ∼2.0 nm indicate HEWL monomer, while those at 12.5 and 17.4 nm along with shoulders at R_h ∼9.0 and 28.7 nm reveal multimeric aggregates. Error bars, SD; N  =  3.</p

Morphology of HEWL aggregates.

<p>Observed morphology of HEWL aggregates after incubation in pH 12.2 (unless indicated otherwise) for 12 hours observed using non-contact mode atomic force microscopy with indicated monomer concentrations is shown. <b>A</b>: 3 µM; <b>B</b>: 120 µM; <b>C</b>: 0.3 µM; <b>D</b>: 120 µM; <b>E</b>: 0.3 µM; <b>F</b>: 3 µM in pH 7; <b>G</b>: 0.3 µM; <b>H</b>: fibril in ‘<b>G</b>’ rescanned at higher magnification; Scale bar  =  100 nm. Heights in Z-axis for all images were within 0—2 nm. See images of amyloid fibrils after 3 weeks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087256#pone.0087256.s004" target="_blank">Figure S4</a>.</p

Schematic model for isodesmic aggregation of HEWL at pH 12.2.

<p>Exposure to pH(∼0.3 µM), moderate (3—10 µM) and high (120 µM) equilibrium aggregation intermediates or protein nanoparticles of increasing average size were obtained. <i>Step A</i> highlights formation of intermolecular disulphide bonds (thick bars) after 100 hours in pH 12.2. <i>Step B</i> yields stable polycationic HEWL nanostructures after dilution into 100 mM pH 7 sodium phosphate buffer after 134 hours in pH 12.2. Thin line clusters indicate amyloid fibrils formed on pathway.</p

Binding of ANS to HEWL aggregates at equal concentration.

<p>Changes in total ANS fluorescence intensity with time after dilution of 144 (a) and 264 hours (b) incubated 120 µM (filled circles) and 40 µM (unfilled circles) HEWL samples in pH 12.2 into 100 mM sodium phosphate buffer at pH 7 are shown. To facilitate comparison, HEWL concentration interacting with ANS in cuvette was maintained constant at 4 µM in both (a) and (b). Error bars, SD; N  =  2.</p

Stability of HEWL aggregates transferred to pH 7.

<p><b>a,</b> Changes in dansyl r_ss after 10-fold dilution of 12, 24, 110 and 134 hours incubated 120 µM HEWL sample in pH 12.2 to 100 mM sodium phosphate buffer at pH 7 are shown. <b>b,</b> Changes in r_ss after a similar dilution with 40 µM HEWL sample is shown. <b>c,</b> Changes in total ANS fluorescence intensity after 10-fold dilution of 12, 24, 72, 120 and 156 hours incubated 120 µM HEWL sample in pH 12.2 into 100 mM sodium phosphate buffer at pH 7 are shown.<b>d,</b> Changes in total ANS fluorescence intensity after a similar dilution with 40 µM HEWL sample is shown. Error bars, SD; N  =  3.</p

Challenges in Accessing and Delivering Maternal and Child Health Services during the COVID-19 Pandemic: A Cross-Sectional Rapid Survey from Six States of India

Background/Objectives: Globally, the COVID-19 pandemic and its prevention and control policies have impacted maternal and child health (MCH) services. This study documents the challenges faced by patients in accessing MCH services, and the experiences of health care providers in delivering those services during the COVID-19 outbreak, explicitly focusing on the lockdown period in India. Methods: A cross-sectional study (rapid survey) was conducted in 18 districts from 6 states of India during March to June, 2020. The sample size included 540 MCH patients, 18 gynaecologists, 18 paediatricians, 18 district immunisation officers and 108 frontline health workers. Bivariate analysis and multivariable analysis were used to assess the association between sociodemographic characteristics, and challenges faced by the patients. Results: More than one-third of patients (n = 212; 39%) reported that accessing MCH services was a challenge during the lockdown period, with major challenges being transportation-related difficulties (n = 99; 46%) unavailability of hospital-based services (n = 54; 23%) and interrupted outreach health services (n = 39; 18.4%). The supply-side challenges mainly included lack of infrastructural preparedness for outbreak situations, and a shortage of human resources. Conclusions/Recommendations: A holistic approach is required that focuses on both preparedness and response to the outbreak, as well reassignment and reinforcement of health care professionals to continue catering to and maintaining essential MCH services during the pandemic