Mn Single-Atom Nanozyme Functionalized 3D-Printed Bioceramic Scaffolds for Enhanced Antibacterial Activity and Bone Regeneration.

Infective bone defect is increasingly threatening human health. How to achieve the optimal antibacterial activity and regenerative repair of infective bone defect simultaneously is a huge challenge in clinic. Herein, this work reports a rational integration of Mn single-atom nanozyme into the 3D-printed bioceramic scaffolds (Mn/HSAE@BCP scaffolds). The integrated Mn/HSAE@BCP scaffolds can catalyze the conversion of H2O2 to produce hydroxyl radical (•OH) and superoxide anion (O2 •-) through cascade reaction. Besides, the prominent thermal conversion efficiency of Mn/HSAE@BCP scaffolds can be utilized for sonodynamic therapy (SDT). The synergetic strategy of chemodynamic therapy (CDT)/SDT enables the sufficient generation of reactive oxygen species (ROS) to kill Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli). Furthermore, the enhanced antibacterial efficacy of Mn/HSAE@BCP scaffolds is beneficial to upregulate the expression of osteogenesis-related markers (such as collagen 1(COL1), Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteoprotegerin (OPG)) in vitro and further promote bone regeneration in vivo. The results demonstrate the good potential of Mn/HSAE@BCP scaffolds for the enhanced antibacterial activity and bone regeneration, which provide an effective method for the treatment of clinical infective bone defect.

Limited value of platelet-related markers in diagnosing periprosthetic joint infection.

OBJECTIVE: To evaluate the diagnostic values of serum platelet count (PC), mean platelet volume ratio (MPV), platelet count to mean platelet volume ratio (PVR), platelet to lymphocyte ratio (PLR), platelet to neutrophil ratio (PNR), PC/Albumin-globulin ratio (PC/AGR), and PC/C-reactive protein (PC/ CRP) in the diagnosis of periprosthetic joint infection (PJI). METHODS: The medical records were retrospectively analyzed of the 158 patients who had undergone hip or knee revisions from January 2018 to May 2022. Of them, 79 cases were diagnosed with PJI and 79 with aseptic loosening (AL). PJI was defined using the Musculoskeletal Infection Society criteria. The plasma levels of CRP, the erythrocyte sedimentation rate (ESR), PC, MPV, PVR, PLR, PNR, PC/AGR, and PC/CRP in the 2 groups were recorded and analyzed. In addition, tests were performed according to different joint types. The receiver operating characteristic curve was used to calculate the sensitivity and specificity of each indicator. The diagnostic value for each indicator was calculated according to the area under the curve (AUC). RESULTS: The PC, PVR, PLR and PC/AGR levels in the PJI group were significantly higher than those in the AL group, while PC/CRP levels were significantly lower (P < 0.001). The AUC for PC/CRP, and PC/AGR was 0.804 and 0.802, respectively, which were slightly lower than that of CRP (0.826) and ESR (0.846). ROC analysis for PC/CRP, and PC/AGR revealed a cut-off value of 37.80 and 160.63, respectively, which provided a sensitivity of 73.42% and 84.81% and a specificity of 75.95% and 65.82% for PJI. The area under the curve of PLR and PC was 0.738 and 0.702. The area under the curve values for PVR, PNR, and MPV were 0.672, 0.553, and 0.544, respectively. CONCLUSIONS: The results of this study suggest that PC, PLR, PC/CRP, and PC/AGR values do not offer significant advantages over ESR or CRP values when employed for the diagnosis of PJI. PVR, PNR, and MPV were not reliable in the diagnosis of PJI.

An Exciting Performance of Established and Novel Biomarkers in Diagnosing Periprosthetic Joint Infections: A Single-center Retrospective Cohort Study.

OBJECTIVE: Significant progress has been made in recent years in the diagnosis of periprosthetic joint infections (PJI). However, the lack of a gold standard test for the diagnosis of PJI remains a challenge.The aim of this study was to evaluate the diagnostic values of the albumin/fibrinogen ratio (AFR), the C-reactive protein/albumin ratio (CAR), and the levels of fibrinogen (FIB) and albumin (ALB) in the diagnosis of PJI. METHODS: The medical records of 158 patients who had undergone hip or knee revisions from January 2018 to May 2022 were retrospectively analyzed. Of these patients, 79 were diagnosed with PJI, while 79 were diagnosed with aseptic loosening (AL). PJI was defined using the Musculoskeletal Infection Society criteria. The plasma levels of C-reactive protein (CRP), ALB, and FIB; the erythrocyte sedimentation rate (ESR); and the AFR and CAR in the two groups were recorded and analyzed. The receiver operating characteristic curve was used to calculate the sensitivity and specificity of each indicator; the diagnostic value for each indicator was calculated as the area under the curve (AUC). RESULTS: The ESR, CRP, FIB, and CAR values in the PJI group were significantly higher than those in the AL group, and the ALB and AFR values were significantly lower than those in the AL group (p < 0.001). The AUC values of AFR and fibrinogen were 0.851 and 0.848, respectively, which were slightly higher than those of CRP (0.826) and ESR (0.846). The AUC of CAR was 0.831 which was slightly lower than that of CRP (0.846). ALB had an AUC of 0.727. The optimal threshold, sensitivity, and specificity, respectively, were 10.05, 84.81%, and 82.28% for AFR; 4.03 µg/mL, 77.22%, and 86.08% for FIB; 0.23, 72.15%, and 82.28% for CAR; and 37.30 g/L, 65.82%, and 73.42% for ALB. CONCLUSIONS: AFR, CAR, and FIB are good new auxiliary diagnostic indicators of PJI, while ALB is of fair value for the diagnosis of PJI.

Recent Progress in Developing a LiOH-Based Reversible Nonaqueous Lithium-Air Battery.

The realization of practical nonaqueous lithium-air batteries (LABs) calls for novel strategies to address their numerous theoretical and technical challenges. LiOH formation/decomposition has recently been proposed as a promising alternative route to cycling LABs via Li2 O2 . Herein, the progress in developing LiOH-based nonaqueous LABs is reviewed. Various catalytic systems, either soluble or solid-state, that can activate a LiOH-based electrochemistry are compared in detail, with emphasis in providing an updated understanding of the oxygen reduction and evolution reactions in nonaqueous media. We identify the key factors that can switch the cell chemistry between Li2 O2  and LiOH and highlight the debate around these routes, as well as rationalize potential causes for these opposing opinions. The identities of the reaction intermediates, activity of redox mediators and additives, location of reaction interfaces, causes of parasitic reactions, as well as the effect of CO2  on the LiOH electrochemistry, all play a critical role in altering the relative rates of a series of interconnected reactions and all warrant further investigation.

Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin.

We aimed to investigate the effect of the genetic mutant G71R (c. 211G > A) in uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) on the glucuronidation of unconjugated bilirubin. The UGT1A1 wild-type and mutant G71R gene sequences were inserted into the lentiviral vector GV358 plasmid and then transfected into COS-7 cells. Real-time polymerase chain reaction and western blot analyses were used to determine mRNA and protein expression levels of UGT1A1, respectively. High-performance liquid chromatography was used to quantitate the levels of conjugated bilirubin. The results showed no significant difference in the mRNA and protein expression levels between the UGT1A1 wild-type and G71R homozygous and heterozygous mutants. The level of conjugated bilirubin reached a maximum in wild-type UGT1A1-transfected COS-7 cells. However, relative to the UGT1A1 wild-type, conjugated bilirubin concentrations were 71 and 22% with G71R heterozygous- and G71R homozygous-transfected COS-7 cells, respectively. In conclusion, we successfully established in vitro cell models of the UGT1A1 wild-type and the G71R homozygous and heterozygous mutants using a lentiviral vector. Furthermore, the catalytic activity for unconjugated bilirubin was lower in the mutant G71R than the UGT1A1 wild-type enzyme, and a weaker effect was observed in the homozygote.

Unraveling the Reaction Interfaces and Intermediates of Ru-Catalyzed LiOH Decomposition in DMSO-Based Li-O2 Batteries.

Investigation of LiOH decomposition in nonaqueous electrolytes not only expands the fundamental understanding of four-electron oxygen evolution reactions in aprotic media but also is crucial to the development of high-performance lithium-air batteries involving the formation/decomposition of LiOH. In this work, we have shown that the decomposition of LiOH by ruthenium metal catalysts in a wet DMSO electrolyte occurs at the catalyst-electrolyte interface, initiated via a potential-triggered dissolution/reprecipitation process. The in situ UV-vis methodology devised herein provides direct experimental evidence that the hydroxyl radical is a common reaction intermediate formed in several nonaqueous electrolytes; this method is applicable to study other battery systems. Our results highlight that the reactivity of the hydroxyl radical toward nonaqueous electrolyte represents a major factor limiting O2 evolution during LiOH decomposition. Coupling catalysts restraining hydroxyl reactivity with electrolytes more resistant to hydroxyl radical attack could help improve the reversibility of this reaction.

Coupling Water-Proof Li Anodes with LiOH-Based Cathodes Enables Highly Rechargeable Lithium-Air Batteries Operating in Ambient Air.

Realizing an energy-dense, highly rechargeable nonaqueous lithium-oxygen battery in ambient air remains a big challenge because the active materials of the typical high-capacity cathode (Li2 O2 ) and anode (Li metal) are unstable in air. Herein, a novel lithium-oxygen full cell coupling a lithium anode protected by a composite layer of polyethylene oxide (PEO)/lithium aluminum titanium phosphate (LATP)/wax to a LiOH-based cathode is constructed. The protected lithium is stable in air and water, and permits reversible, dendrite-free lithium stripping/plating in a wet nonaqueous electrolyte under ambient air. The LiOH-based full cell reaction is immune to moisture (up to 99% humidity) in air and exhibits a much better resistance to CO2 contamination than Li2 O2 , resulting in a more consistent electrochemistry in the long term. The current approach of coupling a protected lithium anode with a LiOH-based cathode holds promise for developing a long-life, high-energy lithium-air battery capable of operating in the ambient atmosphere.

Diagnostic Value of MRI Lamellated Hyperintense Synovitis in Periprosthetic Infection of Hip.

OBJECTIVE: To investigate the correlation between magnetic resonance imaging (MRI) lamellated hyperintense synovitis and periprosthetic infection of hip arthroplasty and estimate its value in the diagnosis of infection after hip replacement. METHODS: A retrospective analysis of 50 patients who underwent MRI from January 2016 to June 2019 after hip replacement was performed. The MRI scanning was performed with a 1.5T clinical imaging unit using SEMAC protocols. A total of 25 patients (cohort 1) showed infected total hip arthroplasty, and 25 patients had non-infected arthroplasty as controls (cohort 2). Two musculoskeletal radiologists, blinded to the clinical diagnosis, reviewed all the images for the presence of lamellated hyperintense synovitis independently. The cases were rereviewed by each reader after 2 weeks. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the first reads. The Kappa statistic was used to assess inter-observer and intra-observer reliability. RESULTS: The incidence of lamellated hyperintense synovitis was 76%-88% in the experimental group and 8%-16% in the control group. The sensitivity of lamellated hyperintense synovitis for infection was 0.80-0.88 (95% confidence interval [CI]:0.59 - 0.97), the specificity was 0.84~0.92 (95% CI: 0.64 -0.99), the positive predictive value 0.83-0.92 (95% CI: 0.67 - 0.98), the negative predictive value 0.81 - 0.88 (95% CI: 0.65 - 0.96). The agreement between two readers was substantial (Kappa = 0.76, 95% CI: 0.58 - 0.94, P < 0.05). There were moderate inter-observer agreements for both readers, reader 1 (Kappa = 0.48, 95%CI: 0.23 - 0.72, P < 0.05) and reader 2 (Kappa = 0.44，95% CI: 0.19 - 0.69, P < 0.05). CONCLUSION: In this cohort, the presence of lamellated hyperintense synovitis in the MRI of hip arthroplasty showed high sensitivity and specificity for infection. This sign had substantial intra-observer reliability and moderate inter-observer reliability in the classification of the synovial pattern.

Expression levels of IL-15 and IL-17 in synovial fluid of rheumatoid arthritis animal model.

The aim of the present study was to investigate the expression levels of interleukin-15 (IL-15) and interleukin-17 (IL-17) in synovial fluid of rheumatoid arthritis (RA) animal model, and to investigate their correlations with RA. A total of 100 Wistar rats were selected, among which 60 rats were used to establish the collagen II-induced arthritis (CIA) model as the model observation group, and the remaining 40 rats were used as blank control group. The levels of IL-15 and IL-17 in synovial fluid were detected via enzyme-linked immunosorbent assay (ELISA) at 1, 7, 14, 21 and 28 days after successful modeling. RA was evaluated by using arthritis index (AI) and pedal swelling volume. The expression levels of IL-15 and IL-17 in synovial fluid of rats in model observation group were higher than those in blank control group (P<0.05), and the levels of IL-15 and IL-17 in model observation group were gradually increased over time. In model observation group at 7 days after modeling, AI and pedal swelling volume began to be increased gradually reaching a peak at 28 days. The pedal swelling volume of CIA model rats was significantly higher than that of the blank control group (P<0.05). The increased expression levels of IL-15 and IL-17 in synovial fluid of rats in the CIA model observation group are correlated with the activity of disease, which can be used as reference indexes for the activity of RA.

The efficacy and safety of methylprednisolone for pain control after total knee arthroplasty: A meta-analysis of randomized controlled trials.

BACKGROUND: Pain management after arthroplasties has become a serious problem. We perform a meta-analysis from randomized controlled trial (RCTs) to examine the efficacy and safety of methylprednisolone in the setting of postoperative pain after total knee arthroplasty (TKA). METHODS: We conduct electronic searches of Medline (1966 to October 2017), Embase (1980 to October 2017), ScienceDirect (1985 to October 2017) and the Web of Science (1995 to October 2017). Eligibility criteria: (1) Participants: Published studies enrolling adult human subjects with knee osteoarthritis who prepare for TKA are included in our study; (2) Interventions: The intervention groups receive methylprednisolone in the management of postoperative pain; (3) Comparisons: The control groups receive placebo; (4) Outcomes: The primary outcomes are visual analogue scale (VAS) scores and narcotic consumption. The secondary outcomes include adverse effects (gastrointestinal events and pruritus) and postoperative complications (deep venous thrombosis, pulmonary embolism and infection); (5) Study design: RCTs. Fixed/random effect model is adopted according to the heterogeneity tested by I2 statistic. Stata 11.0 software is used to perform the meta-analyses. RESULTS: Four RCTs involving 248 patients are included. The present meta-analysis indicates that there are significant differences between groups regarding pain score at 6 h (WMD = -0.661, 95% CI: -1.152 to -0.171, P = 0.008), 12 h (WMD = -0.555, 95% CI: -1.091 to -0.018, P = 0.043) and 24 h (WMD = -0.648, 95% CI: -1.146 to -0.150, P = 0.011) after TKA. Significant differences are found in terms of narcotic consumption at 6 h (WMD = -3.453, 95% CI: -6.116 to -0.791, P = 0.011), 12 h (WMD = -5.138, 95% CI: -9.036 to -1.240, P = 0.010) and 24 h (WMD = -3.651, 95% CI: -5.909 to -1.393, P = 0.002). CONCLUSION: Methylprednisolone could significantly decrease postoperative pain score, narcotic consumption and opioid-related adverse effects after TKA. Further high-quality RCTs are still required to validate the results.

UGT1A1 gene mutations and neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations.

BACKGROUND: Uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene mutation was shown to be responsible for neonatal hyperbilirubinemia. This study aimed to investigate whether UGT1A1 gene mutation is associated with neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. METHODS: Two hundred and eighteen infants with hyperbilirubinemia (118 Heiyi Zhuang, 100 Han) and 190 control subjects (110 Heiyi Zhuang, 80 Han) were enrolled. Polymerase chain reaction and gene sequencing were used to detect the TATA-box and exon 1 of UGT1A1. RESULTS: (TA)7 insertion mutation, 211G>A (G71R), 686C>A (P229Q), and 189C>T (D63D) were detected. Logistic regression analysis showed odds ratios (OR) of 2.64 (95% confidence interval (CI) 1.64-4.24; P < 0.001) and 0.69 (95%CI 0.43-1.10; P = 0.115) for neonates who carried UGT1A1 G71R and (TA)7 insertion mutation, respectively. G71R homozygosity increased the odds of dangerous bilirubin levels by a factor 34.23, and G71R heterozygosity only by 2.10. CONCLUSION: We found that UGT1A1 G71R mutation is a risk factor for neonatal hyperbilirubinemia in Guangxi Heiyi Zhuang and Han populations. Meanwhile, the UGT1A1 (TA)7 insertion mutation is not associated with neonatal hyperbilirubinemia in the two ethnic groups.

[Effect and mechanism of high-dose Vitamin B3 on granulopoiesis in normal rat].

OBJECTIVE: To investigate the effect and mechanism of high dose Vitamin B3 on granulopoiesis in normal rat. METHODS: Twenty one healthy SD rats were randomly divided into three groups: the Vitamin B3 group (Vit B3 500 mg·kg⁻¹·d⁻¹, × 7 d), the rhG-CSF group (rhG-CSF 25 µg·kg⁻¹·d⁻¹, × 7 d) and the normal saline group (2 ml/d, × 7 d). The peripheral blood cell counts were analyzed by automatic blood cell counter before (day 0) treatment, the third day (day 3) and the seventh day (day 7) after administration of drugs, respectively. The concentration of serum nicotinamide adenine dinucleotide (NAD⁺) level was measured by enzymatic cycling assay before and after drugs treatment. The expressions of G-CSF, G-CSFR, SIRT1, C/EBPα, C/EBPß, C/EBPε and NAMPT mRNA were detected by reverse transcription real-time fluorescent quantitative PCR. RESULTS: The neutrophil counts increased significantly after 7 days of Vitamin B3 and rhG-CSF treatment compared with that of control group [(1.64 ± 0.19) × 109/L, (1.88 ± 0.37)× 109/L vs (0.86 ± 0.18) × 109/L, P<0.01]; the level of serum NAD⁺ increased significantly [(0.96 ± 0.08) nmol/L, (0.65 ± 0.12) nmol/L vs (0.36 ± 0.15) nmol/L, P<0.01]; the expression of G-CSF, G-CSFR, SIRT1, C/EBPα, C/EBPε and NAMPT mRNA in bone marrow mononuclear cells were increased significantly compared with that of control group (P<0.01). CONCLUSION: High dose of Vitamin B3 may play an important role in increasing absolute neutrophil count in healthy rat under steady state, and the mechanism may be dependent on NAMPT-NAD⁺-SIRT1 signaling pathways.

[Surgical approaches for intertrochanteric fractures in elder patients].

OBJECTIVE: To conduct long-term follow-ups on the efficacies of surgical approaches for intertrochanteric fractures in elder patients. METHODS: The outcomes of intertrochanteric fractures in 402 elder patients undergoing different surgical procedures during 2005 to 2009 were reviewed. The modified classification of Singh index, detailed surgical contraindications and improved functional scoring system specialized for intertrochanteric fracture were included. Among these, type I to IV had 17 cases (4%), 49 cases (12%), 187 cases (72%), 48 cases (12%). The follow-up period was 1 - 5 years. The orthopedic complications, rehabilitation duration and functional scores were reviewed. RESULTS: Most patients achieved self-care at 1 month post-operation. The failure rate of internal fixation was 5% and infection rate 1.9% in hemiarthroplasty. type I to IV had 70.6% and 100.0%, 85.7% and 86.7%, 85.4% and 92.0%, 72.9% and 87.5% cases showed excellent or good outcomes at 3 month and 1 year. During a follow-up period of 5 years, there was no occurrence of prothetic loosening or excessive wear of acetabular. CONCLUSION: A rational selection of surgical approaches may markedly reduce the failure rate of internal post-operative fixation and shorten the rehabilitation period. And the long-term follow-up outcomes are satisfactory.

[Roles of UGT 1A1 gene mutation in the development of neonatal hyperbilirubinemia in Guangxi].

OBJECTIVE: Neonatal unconjugated hyperbilirubinemia is one of the most common conditions encountered by the practicing pediatricians. Although it is usually self-limited and benign, the condition is of importance because of the rare instances in which severe hyperbilirubinemia can lead to bilirubin encephalopathy or kernicterus. The uridine diphosphate-glucuronosyl transferase 1A1 (UGT 1A1) gene controls bilirubin conjugation by determining the structure of the enzyme glucuronosyltransferase, which is synthesized in the hepatocyte. In the recent years much has been learned about the relationship between UGT 1A1 gene mutation and neonatal hyperbilirubinemia. This study aimed to investigate the roles of UGT 1A1 gene mutation in the development of neonatal hyperbilirubinemia in Guangxi. METHODS: A total of 73 cases with hyperbilirubinemia and 31 healthy neonates were enrolled. UGT 1A1 G71R genotypes were identified by the (amplification refractory mutation system, ARMS) and direct sequencing method in all the neonates. To analyze the incidence of bilirubin encephalopathy, the peak (total serum bilirubin, TSB) concentration after 72 hours of age, and the possibility of TSB > 20 mg/dl of each group. RESULTS: (1) The frequencies of allele G71R were 0.1915 in this study, 0.2329 in hyperbilirubinemia group vs. 0.097 in healthy groups. The allele gene frequency of G71R in neonatal hyperbilirubinemia was higher than that in the normal group (P < 0.05). (2) Homozygous neonates had higher possibility to develop bilirubin encephalopathy and higher TSB concentration 72 hours after birth (28.57%, 23.12 ± 4.58) than the normal group (0%, 17.68 ± 2.69). The difference between the former two was significant (P < 0.001). (3) The TSB of the 5 neonates was > 20 mg/dl in G71R homozygous type, the odds ratio and 95%CI were 7.955 (1.349, 46.899). CONCLUSION: (1) G71R mutation gene was associated with neonatal jaundice in Guangxi region. (2) The possibility of TSB > 20 mg/dl in G71R homozygous was higher than those of the wild-type. (3) The incidence of bilirubin encephalopathy and TSB concentration after 72 hours of age for neonates who were homozygous to G71R gene were higher than the wild-type.

[Relationship between glucose-6-phosphate dehydrogenase gene mutations and neonatal jaundice in Naning, Guangxi].

OBJECTIVE: To study the correlation between glucose-6-phosphate dehydrogenase (G-6-PD) activities and three common mutations of G-6-PD gene G1388A, G1376T and A95G and investigate the effects of G-6-PD gene mutations on neonatal jaundice in Nanning, Guangxi. METHODS: One hundred and twenty-four neonates from Nanning, Guangxi, with hyperbilirubinemia were enrolled. The ARMS-PCR and PCR/REA methods were used to determine G-6-PD gene mutations. G-6-PD activities were measured using the NBT method. The incidence of acute bilirubin encephalopathy and the peak bilirubin concentration 72 hrs after birth were compared between the neonates with different genotypes and between the G-6-PD mutation and normal groups. The risk of blood serum bilirubin >340 mumol/L was evaluated by logistic regression analysis. RESULTS: Of the 124 cases, gene mutations were found in 37 cases, including G1388A (n=20), G1376T (n=14), A95G (n=4) and G1388A+A95G (n=1). Five cases (25%) showed normal G-6-PD activities in the G1388A gene mutation group and 4 (29%) had normal G-6-PD activities in the G1376T G1388A gene mutation group. All of 4 cases of A95G G1388A gene mutation showed a deficiency of G-6-PD activities. There were no significant differences in the incidence of acute bilirubin encephalopathy and the peak bilirubin concentration 72 hrs after birth between the G1388A and G1376T G1388A gene mutation groups. The incidence of acute bilirubin encephalopathy, the peak bilirubin concentration 72 hrs after birth and the risk of serum bilirubin >340 micromol/L in the G-6-PD mutation group were not different from the normal group. CONCLUSIONS: G1388A, G1376T and A95G are common G-6-PD gene mutations in Nanning, Guangxi. The false negative results may be received when the NBT method is used for diagnosis of G-6-PD deficiency. There are similar effects on the incidence of acute bilirubin encephalopathy and the peak bilirubin concentration 72 hrs after birth between different gene mutation groups. G-6-PD gene mutations alone may not contribute to the development of acute bilirubin encephalopathy and the changes of peak bilirubin concentration 72 hrs after birth and the risk of serum bilirubin >340 micromol/L.