Transglutaminases

The plasmid pNL4-3_V1Q3_V4A1 ΔRT encoding for HIV-1_NL4-3 Env and plasmid Q23_BG505_V1Q3-V4A1 ΔRT encoding for HIV-1_BG505 Env carrying the Q3 (GQQQLG) and A1 (GDSLDMLEWSLM) labeling peptides (Wu et al., 2006 (link); Yin et al., 2006 (link); Zhou et al., 2007 (link)) in V1 and V4 loops of full-length pNL4-3 ΔRT construct and full-length Q23_BG505 ΔRT construct, respectively; and the Env-expressing plasmid pCAGGS_JR-FL_V1Q3_V4A1 containing the peptides in analogous positions were previously described (Munro et al., 2014 (link)). The D368R point mutation (Olshevsky et al., 1990 (link)) was introduced into untagged constructs and V1Q3_V4A1 tagged constructs of full-length NL4-3, BG505 and Env-expressing JR-FL by overlap-extension PCR. All viruses were produced by co-transfecting HEK293 cells using the following ratios of plasmids:
HIV-1_NL4-3: 40:1 of pNL4-3 ΔRT to pNL4-3_V1Q3_V4A1 ΔRT;
HIV-1_BG505: 40:1 of Q23_BG505 ΔRT to Q23_BG505_V1Q3_V4A1 ΔRT;
HIV-1_JR-FL: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL to pCAGGS_JR-FL_V1Q3_V4A1.
HIV-1_NL4-3_D368R: 40:1 of pNL4-3_D368R ΔRT to pNL4-3_V1Q3_V4A1_D368R ΔRT.
HIV-1_BG505_D368R: 40:1 of Q23_BG505_D368R ΔRT to Q23_BG505_V1Q3_V4A1_D368R ΔRT.
HIV-1_JR-FL_D368R: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL_D368R to pCAGGS_JR-FL_V1Q3_V4A1_D368R.
HIV-1_NL4-3 mixed trimer 1: 40:1 of pNL4-3_D368R ΔRT to pNL4-3_V1Q3_V4A1 ΔRT.
HIV-1_BG505 mixed trimer 1: 40:1 of Q23_BG505_D368R ΔRT to Q23_BG505_V1Q3_V4A1 ΔRT.
HIV-1_JR-FL mixed trimer 1: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL_D368R to pCAGGS_JR-FL_V1Q3_V4A1.
HIV-1_NL4-3 mixed trimer 2: 20:20:1 of pNL4-3 ΔRT to pNL4-3_D368R ΔRT to pNL4-3_V1Q3_V4A1_D368R ΔRT.
HIV-1_BG505 mixed trimer 2: 20:20:1 of Q23_BG505 ΔRT to Q23_BG505_D368R ΔRT to Q23_BG505_V1Q3_V4A1_D368R ΔRT.
HIV-1_JR-FL mixed trimer 2: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 20:20:1 for pCAGGS_JR-FL to pCAGGS_D368R to pCAGGS_JR-FL_V1Q3_V4A1_D368R.
Statistically, the 20:20:1 ratio for mixed trimer 2 will yield 50% trimers with the desired mixed trimer two shown in Figure 2B.
HIV-1_NL4-3 mixed trimer 3: 40:1 of pNL4-3 ΔRT to pNL4-3_V1Q3_V4A1_D368R ΔRT.
HIV-1_BG505 mixed trimer 3: 40:1 of Q23_BG505 ΔRT to Q23_BG505_V1Q3_V4A1_D368R ΔRT.
HIV-1_JR-FL mixed trimer 3: 1:1 of the pNL4-3 ΔEnvΔRT and the Env-expressing constructs, which were at 40:1 for pCAGGS_JR-FL to pCAGGS_JR-FL_V1Q3_V4A1_D368R.
Virus was harvested 40 hr post transfection and concentrated by centrifugation over a 15% sucrose cushion at 20,000 x g for 2 hr. Virus pellets were resuspended in the labeling buffer containing 50 mM HEPES, 10 mM MgCl2, 10 mM CaCl2. Resuspended virus was enzymatically labeled with fluorophores in a reaction mixture containing Cy3B(3S)-cadaverine (0.5 μM), transglutaminase (0.65 μM; Sigma Aldrich), LD650-CoA (0.5 μM) (Lumidyne Technologies), and AcpS (5 μM) (Zhou et al., 2007 (link)). The labeling reaction was incubated at room temperature overnight. 0.1 mg/ml PEG2000-biotin (Avanti Polar Lipids) was added to the labeling reaction and incubated for 30 min at room temperature. Under these conditions, the labeling efficiencies of the Q3 and A1 tags are 40% and 55%, respectively (Munro et al., 2014 (link)). The virus was then purified away from excess fluorophore and lipid by ultracentrifugation (1 hr at 150,000 x g) over a 6–18% Optiprep (Sigma Aldrich) gradient containing 50 mM Tris pH 7.4 and 50 mM NaCl. The fractions containing viral particles were identified by p24 western blot and stored at −80°C.

Blood samples were collected from the antecubital vein of all participants in the morning under fasting conditions. They were stored in vacuum tubes containing EDTA (ethylene diamine tetraacetic acid) and coagulation tubes. A range of haematological and biochemistry tests (Table 2) were conducted on fresh samples at the central laboratory of the Staff Hospital of Jidong oil-field of Chinese National Petroleum. Fasting blood glucose was measured with the hexokinase/glucose-6-phosphate dehydrogenase method. Cholesterol and triglyceride concentrations were determined by enzymatic methods (Mind Bioengineering Co. Ltd, Shanghai, China). Blood samples were also measured using an auto-analyzer (Hitachi 747; Hitachi, Tokyo, Japan) at the central laboratory of the Staff Hospital of Jidong oil-field of Chinese National Petroleum. For all participants, serum creatinine, cholesterol, high-density lipoproteins (HDL-C), low-density lipoproteins (LDL-C), triglycerides and glucose levels were assessed. In subgroup analysis studies, various biomarkers of blood cells, serum and plasma were measured: C-reactive protein, homocysteine, estrogens, androgens, vitamin D, lipoprotein-associated phospholipase A2 (Lp-PLA2), insulin, and glycosylated hemoglobin HbA1c.Table 2

Haematology, biochemistry and biological specimen banking in the COACS

	Analysate
Red blood cells	Haemoglobin
	Red corpuscle count
	Haematocrit
	Mean corpuscular volume
	Mean corpuscular
	Haemoglobin concentration
	Red blood cell distribution width
White blood cells	White cell count Total count
	Differential count
Platelets	Platelets Count
	Mean platelet volume
Urea	Urine specific gravity
	Ery
	Urea nitrogen
	Uric acid (UA)
	Creatinine (Cr)
	Urine protein
Liver function tests (plasma)	Alkaline phosphatise
	Alanine transaminase (ALT)
	Aspartate aminotransferase (AST)
	Phosphatise Transglutaminase (TG)
Liver function tests (serum)	HBsAg
	Anti-HBs
	HBeAg
	Anti-HBe
	Anti-HBc
Lipids (plasma)	Total cholesterol (TC)
	Total bilirubin (TBIL)
	Triglycerides (TG)
	Low density lipoprotein (LDL)
	Very Low density lipoprotein (VLDL)
General chemistry (plasma)	C-reactive protein
	Homocysteine
	Steroids
	Glucose
	Insulin
	Glycosylated hemoglobin
Bio-specimen banking
White blood cells	DNA, RNA extraction and analyses
Serum	Pedtidome profiling
Plasma	Glycome

Blood samples were processed and separated onsite for biospecimen banking (−80 °C). DNA and RNA were extracted and stored in the laboratory of Beijing Key Laboratory of Clinical Epidemiology, Beijing, China.

The ECM is comprised of a network of gelatin and fibrin. To prepare the ECM components, a 15 wt/v% gelatin solution (Type A, 300 bloom from porcine skin, Sigma) is first produced by adding gelatin powder to a warm solution (70 °C) of DPBS (1X Dulbelco’s phosphate buffered saline without calcium and magnesium). The gelatin is allowed to fully dissolve by stirring for 12 h at 70 °C, and the pH is then adjusted to 7.5 using 1 M NaOH. The solution is sterile filtered and stored at 4 °C in aliquots for later usage in casting (<3 months). A fibrinogen solution (50 mg/mL) is produced by dissolving lyophilized bovine blood plasma protein (Millipore) at 37 °C in sterile DPBS without calcium and magnesium. The solution is held at 37 °C without agitation for at least 45 min to allow complete dissolution. The transglutaminase (TG) solution (60 mg/mL) is prepared by dissolving lyophilized powder (Moo Gloo) in DPBS without calcium and magnesium and gently mixing for 20 sec. The solution is then held at 37 °C for 20 min and sterile filtered before using. A CaCl₂ stock solution (250 mM) is prepared by dissolving CaCl₂ powder in DPBS without calcium and magnesium (Corning). To prepare stock solution of thrombin, lyophilized thrombin (Sigma Aldrich) is reconstituted at 500 U/mL using sterile DPBS and stored at −20 °C. Thrombin aliquots are thawed immediately prior to use.
A controlled stress rheometer (DHR-3, TA Instruments, New Castle, DE) with a 40 mm diameter, 2° cone and plate geometry is used for ink rheology measurements. The shear storage (G′) and loss (G″) moduli are measured at a frequency of 1 Hz and an oscillatory strain (γ) of 0.01. Time sweeps are conducted by rapidly placing a premixed ECM solution that contains thrombin onto the Peltier plate held at 37 °C.