Vitamin K1

The chickens were sacrificed under chloroform anesthesia by cervical dislocation. Whole caeca with their contents originating from 18 random healthy chickens or hens 4 to 40 weeks of age were removed during necropsy, chilled on ice and transported to an anaerobic chamber for further processing within one hour. The caeca were opened in an anaerobic chamber (10% CO₂, 5% H₂ and 85% N₂ atmosphere; Concept 400, Baker Ruskinn, USA) and 0.5 g of content was squeezed into 4.5 ml pre-reduced PRAS dilution blank (0.1 g magnesium sulfate heptahydrate, 0.2 g monobasic potassium phosphate, 0.2 g potassium chloride, 1.15 g dibasic sodium phosphate, 3.0 g sodium chloride, 1.0 g sodium thioglycolate, 0.5 g L-cysteine, 1000 ml distilled water; final pH 7.5 +/− 0.2 at 25 °C) and mixed thoroughly. All samples were serially diluted in pre-reduced PRAS dilution blank and plated on Wilkins-Chalgren anaerobe agar (WCHA) (Oxoid) supplemented with 30% of rumen fluid. The rumen fluid was collected from cows by an oral probe, filtered through cheesecloth, centrifuged at 8000 g for 30 min and sterilised by filtration through a 0.22 μm filter. Aliquots of rumen fluid were stored at − 20 °C. WCHA was additionally supplemented with 5 mg/l hemin, 1 mg/l cellobiose, 0.5 g/l soluble starch, 1 mg/ml maltose, 0.2 ml vitamin K1 solution (0.1 ml of filter sterilized vitamin K1 in 20 ml 95% ethanol) and 0.5 mg/ml L-cysteine. Approx. 10 well-separated colonies of different morphology were selected from each agar plate after a five-day incubation at 37 °C and purified by subculture on WCHA. All isolates were stored at − 80 °C in pre-reduced PRAS dilution blank containing glycerol at 20% concentration and equal volume of sterile sheep blood. Sensitivity of pure anaerobe cultures to air oxygen exposure was tested exactly as described elsewhere [6 (link)]. Briefly, bacterial cultures were serially diluted in anaerobic chamber and plated on 4 copies of WCHA. One copy of WCHA was left in the anaerobic chamber to determine initial counts of each anaerobe. The remaining 3 copies of WCHA plates were placed into a standard aerobic 37 °C incubator and after 1, 8 and 24 h, a single copy of agar plate was returned back to the anaerobic chamber to check for growth restoration.

The atomic coordinates were taken from the X-ray structures; cyanobacterial PSI from Thermosynechococcus elongatus at 2.5 Å resolution (PDB code, ; 1JB0);2 (link) plant PSI from Pisum sativum at 2.8 Å resolution (PDB code, ; 4XK8); PbRC from Rhodobacter sphaeroides at 2.01 Å resolution (PDB code, ; 3I4D), 1.87 Å resolution (PDB code, ; 2J8C),4 (link) and 2.55 Å resolution (PDB code, ; 1M3X);3 (link) PbRC from Thermochromatium tepidum at 2.2 Å resolution (PDB code, ; 1EYS);30 (link) the PSII monomer unit (designated monomer A) of the PSII complexes from Thermosynechococcus vulcanus at 1.9 Å resolution (PDB code, ; 3ARC).5 (link) Hydrogen atoms were generated and energetically optimized with CHARMM.54 Atomic partial charges of the amino acids were adopted from the all-atom CHARMM22) parameter set.55 (link) For PSI, the atomic charges of cofactors were taken from previous studies (Chla, phylloquinone, β-carotene,56 (link) and the Fe₄S₄ cluster57 ). The atomic charges of the other cofactors ((B)Chla, including (B)Chla˙⁺ and (B)Chla˙^–, (B)Pheoa, ubiquinone, plastoquinone, spheroidene, sulfoquinovosyl diacylglycerol, heptyl 1-thiohexopyranoside, and the Fe complex) were determined by fitting the electrostatic potential in the neighborhood of these molecules using the RESP procedure58 (Tables S2–S11†). To obtain the atomic charges of the Mn₄CaO₅ cluster or the Fe complex, backbone atoms are not included in the RESP procedure (except for D1-Ala344) (Table S11†). The electronic wave functions were calculated after geometry optimization by the DFT method with the B3LYP functional and 6-31G** basis sets, using the JAGUAR program.59 For the atomic charges of the non-polar CH_n groups in cofactors (e.g., the phytol chains of (B)Chla and (B)Pheoa and the isoprene side-chains of quinones), the value of +0.09 was assigned for non-polar H atoms. We considered the Mn₄CaO₅ cluster to be fully deprotonated in S₁.
The protein inner spaces were represented implicitly with the dielectric constant ε_w = 80, whereas the following water molecules were represented explicitly; (i) for PSII, ligand water molecules of the Mn₄CaO₅ cluster (W1 to W4), a diamond-shaped cluster of water molecules near TyrZ (W5 to W7)60 (link), the water molecule distal to TyrD44 (link), ligand water molecules of Chl_D1 (A1003 and D424), Chl_D2 (A1009 and A359), and other Chla (B1001, B1007, B1027, C816, and C1004); (ii) for PSI, clusters of water molecules near A_1A (A5007, A5015, A5022, A5043, and A5049) and A_1B (B5018, B5019, B5030, B5055, B5056, and B5058), ligand water molecules of A_–1A (B5005), A_–1B (A5005), and other Chla (A5004, A5010, A5012, A5024, A5032, A5051, B5006, B5010, B5022, B5036, B5053, B5054, J127, L4023, and M155).

The present study is a retrospective cohort study. Patients were divided into two groups: non-survivors and survivors at 3 months. The following variables from medical records were investigated: (1) patient factors (sex, presence of dysphagia, compromised-host, smoking history); (2) clinical findings factors, such as CRP, WBC, blood urea nitrogen (BUN), age, purulence of pleural fluid, infection source (community-acquired/hospital-acquired), serum albumin, OHAT score, and etiology (monomicrobial/polymicrobial/no growth); and (3) treatment methods. Dysphagia was defined as coughing when taking a meal or decreasing swallowing ability on evaluation by physicians and speech-language-hearing therapists [7 (link)]. Data on treatment and outcomes were also evaluated for each patient during hospitalization. A compromised-host was defined as a patient with any of the following diseases: rheumatoid arthritis, chronic kidney disease, malignancy, diabetes, cardiovascular diseases, neurological diseases, and steroid use. We used two clinical risk scores: RAPID (total score; min:0 point, max:7 points) and OHAT (total score; min:0 point, max:16 points). The RAPID score was based on five common parameters (Table 1) [6 (link)]. Based on the results of the dental examinations, the presence of teeth with poor prognosis was retrospectively investigated using panoramic dental radiography. They were defined as teeth with abnormal radiographic findings (e.g., apical radiolucency larger than 3 mm in diameter, alveolar bone loss around more than half of the root, untreated root remnants, or vertically fractured roots) [19 ,20 ]. Medical records were used whether those teeth were extracted. Pleural fluid was collected by pleural puncture at the time of admission, and microbiological examinations were performed. Anaerobic containers were used to collect pleural fluid to detect anaerobic bacteria, and Gram staining and pleural fluid cultures were performed. Blood agar (Kohjin Bio Co., Ltd., Saitama, Japan) and chocolate agar media (Kohjin Bio Co., Ltd.) were used to detect general bacteria. Anaero Columbia agar medium with hemin and vitamin K1 (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) was used to detect anaerobic bacteria; any anaerobic bacteria were then cultivated at 35°C and 9% CO₂. The causative pathogens were then identified in the pleural fluid culture.

The in vitro fermentation was performed based on the previous method with minor modifications (Lam et al., 2018 (link)). First, fresh fecal samples were collected from six healthy volunteers (three women and three men, 20–30 years of age) who maintained a regular diet and had not received antibiotics or prebiotic treatment for 3 months. The fecal samples were immediately mixed with sterilized 0.1 M phosphate-buffered saline (pH 7.0) to produce a 10% (w/v) fecal suspension. Then, 2.0 g of peptone, 2.0 g of yeast extract, 0.5 g of cysteine-HCl, 0.1 g of NaCl, 2.0 g of NaHCO₃, 0.04 g of K₂HPO₄, 0.04 g of KH₂PO₄, 0.01 g of MgSO₄ 7H₂O, 0.01 g of CaCl₂ 6H₂O, 0.02 g of hemin, 0.5 g of bile salt, 2.0 ml of Tween 80, 10 μl of vitamin K₁, and 1.0 ml of resazurin 1% (w/v) were dissolved in 1 L ultrapure water to obtain a basic medium. Finally, LDSPs were selected as 1% (w/v) carbon sources, and the medium without carbon sources and 1% (w/v) inulin were used as a blank control (CON) and positive control (INU). The medium was adjusted to pH 7.0 using 1 mol⁻¹ HCl and placed in an anaerobic chamber at 37°C overnight to pre-reduce the media. Here, 1.0 ml of 10% (w/v) fecal slurry was added to a 9.0 ml medium and placed in the 37°C anaerobic chamber and incubated for 0, 6, 12, and 24 h. Then, the samples were collected immediately and stored at −80°C for further analysis.

Fifteen- to eighteen-week-old male wild-type and Abcg5/Abcg8-deficient mice were anesthetized by isoflurane and administered 25 mg/g body weight of vitamin K₁ via intravenous injection (Nippon Zenyaku Kogyo, Fukushima, Japan). After the vitamin K₁ administration, mice were maintained under anesthesia throughout. One hour after administration, the cystic duct was ligated and a common bile duct fistula was created using a Teflon catheter (UT-03; Unique Medical Co, Ltd., Tokyo, Japan) to collect hepatic bile specimens for 1.5 h. After bile collection, the mice were sacrificed by whole blood sampling followed by liver isolation. All specimens were stored at −80 °C until analysis.
All animal experiments were conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and with protocols approved by the Animal Studies Committee of the University of Tokyo (P17-063).