Biomek 4000

Metabolomics analysis on urine samples was conducted by using the Q300 Metabolite Assay Kit (Human Metabolomics Institute, Inc., Shenzhen, Guangdong, China), referring to reported method with some modifications [21 (link)]. In brief, samples were firstly thawed on the ice-bath to reduce sample degradation. Then, 25 μL of urine was added to a 96-well plate, which was loaded to the Biomek 4000 workstation (Biomek 4000, Beckman Coulter, Brea, California, USA) [21 (link)]. The cold methanol containing partial internal standards was automatically added to each sample, and the samples were subsequently vortexed for 5 min [22 ]. After centrifugation for 30 min at 4000×g (Allegra X-15R, Beckman Coulter, Indianapolis, IN, USA), 30 μL of supernatant and 20 μL of fresh derivative reagents (200 mM 3-NPH in 75% methanol and 96 mM EDC-6% pyridine solution in methanol) were added to each well of a new clean 96-well plate [22 ]. After derivatization at 30 °C for 60 min, each sample was diluted by 350 μL of cold 50% methanol and stored at − 20 °C for 20 min. After centrifugation with the conditions of 4000×g and 4 °C for 30 min, 135 μL of supernatant and 15 μL of internal standards were added to each well on a new 96-well plate. And the remaining wells were filled with serial diluted derivatized stock standards. At last, the sample plate was sealed for the subsequent UPLC-MS analysis.

Small volume 384-well plates (Greiner part 784261) were spotted with 5 μL of each test and control compound, resulting in a 1000× concentration source plate for a 40 nL pin tool (Fig. S1A). For SP assays, libraries were provided preplated at 10 mM in DMSO (MMV). Control compounds primaquine and monensin were plated at 10 mM, MMV390048 (PI4Ki) and atovaquone were plated at 3.3 mM, and nigericin was plated at 200 μM. For DR assays, serial dilutions were made in DMSO (100%) using a Biomek 4000 (Beckman Coulter). Two DR plate maps were used in this study, termed v1 as originally published^{19 (link)} and v2 (Fig. S1B). The assay steps were unchanged regardless of the map version used. Source plates were sealed and shipped on dry ice to Shoklo Malaria Research Unit in Mae Sot, Thailand or Institute Pasteur in Phnom Penh, Cambodia.

We used a liquid-handling robot, Beckman Coulter Biomek 4000, to measure the hydrolytic activity of all of the samples (blank, WT, and F232W/F396W). The samples were measured in triplicate simultaneously in 96-well plates. The purified enzymes were diluted to 100 nm using 100 mm sodium phosphate (pH 6.0) in a low-protein-binding microtube. In 96-well reaction plates, the diluted enzymes were incubated with crystalline chitin (0–6 mg/ml) at 25 °C for 30 min in a reaction mixture volume of 150 μl (1:1 (v/v) enzyme/substrate ratio) without shaking. The reactions were stopped with 200 μl of the Schales' reagent (500 mm sodium carbonate, 1.5 mm potassium ferricyanide). Insoluble chitin was separated on 96-well 1.2-μm hydrophilic low-protein-binding Durapore® membrane filter plates (Merck Millipore). The filtered solution was heated at 95 °C for 15 min, and 100 μl of the samples were transferred to 384-well clear plates. Absorbance at 420 nm was measured using a multimode microplate reader (SpectraMax iD3, Molecular Devices). The amounts of soluble products were calculated from the standard curve with chitobiose. The error bars shown in Fig. 2 (A and B) represent the S.D. values of the sextupled experiments.

LC-MS analyses were performed using an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS) system (ACQUITY UPLC-Xevo TQ-S; Waters Corp., Milford, MA, USA) as previously described^{70 (link)}. Briefly, the freeze-dried fecal samples from 86 PD patients (42 early stage vs. 44 advanced stage) were thawed in an ice-bath, and about 5 mg of each sample was weighted. Then, 20 μL of ultrapure water was added, and 120 μL of methanol containing internal standards solution was added to extract the metabolites. The samples were homogenated for 3 min and centrifuged at 13500 g for 10 min. Then, 30 μL of supernatant was transferred to a 96-well plate for derivatization using the Biomek 4000 workstation (Biomek 4000; Beckman Coulter, Inc., Brea, CA, USA). After derivatization, 400 μL of ice-cold 50% methanol solution was added to dilute the samples. Then, the plate was stored at –20 °C for 20 min and centrifugated at 4000 g for 30 min. Next, 135 μL of supernatant was transferred to a new 96-well plate for LC-MS analysis. The raw data files generated by LC-MS were processed using the MassLynx software (version 4.1; Waters, Milford, MA, USA) to perform peak integration, calibration, and quantitation for each metabolite.

Library preparation and sequencing were performed as described in detail previously, using an automated platform (Biomek4000, Beckman Coulter) [21 (link)]. Briefly, the V3-V4 region of 16S rRNA genes was amplified in duplicates (15 + 10 cycles) following a two-step protocol using primers 341F-785R [22 (link),23 (link)]. After purification using the AMPure XP system, sequencing was carried out with pooled samples in paired-end modus (PE300) using an Illumina MiSeq system according to the manufacturer’s instructions and 25% (v/v) PhiX standard library.

An Agencourt RNAdvance™ Tissue Total RNA Purification Kit (Beckman Coulter Inc., CA, United States) with the aid of Biomek 4000 automated workbench station (Beckman Coulter, Inc., CA, United States, Appendix IV) was used to isolate the total RNA from the samples. The isolated RNA was quantified using NanoDrop 8000 Spectrophotometer (ThermoFisher Scientific, United States). The quality of RNA samples was considered good if the nucleic acid-protein ratio was between 1.9–2.1. Consequently, the complementary DNA (cDNA) was synthesised from 500 ng template RNA using a High-Capacity cDNA Reverse Transcription Kit (Beckman Coulter, Inc., CA, United States) in a 20-μL reaction volume and thermocycling was carried out in a Veriti™ 96-Well Thermal Cycler 7 (Applied Biosystems, California, United States) with the following conditions: 10 min at 25°C, 30 min at 37°C, and 5 min at 95°C.