What Is the Optimal Timing for Reading the Leukocyte Esterase Strip for the Diagnosis of Periprosthetic Joint Infection?

BACKGROUND: The leucocyte esterase (LE) strip test often is used to diagnose periprosthetic joint infection (PJI). In accordance with the manufacturer's directions, the LE strip test result is read 3 minutes after exposing it to joint fluid, but this has not been supported by robust research. Moreover, we have noted that the results of the LE strip test might change over time, and our previous studies have found that centrifugation causes the results of the LE strip test to degrade. Still, there is no evidence-based recommendation as to when to read the LE strip test to maximize diagnostic accuracy, in general, and the best reading times for the LE strip test before and after centrifugation need to be determined separately, in particular. QUESTIONS/PURPOSES: (1) What is the optimal timing for reading LE strip test results before centrifugation to diagnose PJI? (2) What is the optimal timing for reading LE strip test results after centrifugation to diagnose PJI? METHODS: This study was a prospective diagnostic trial. In all, 120 patients who were scheduled for revision arthroplasty and had signs of infection underwent joint aspiration in the outpatient operating room between July 2018 and July 2019 and were enrolled in this single-center study. For inclusion, patients must have had a diagnosis of PJI or nonPJI, valid synovial fluid samples, and must not have received antibiotics within 2 weeks before arthrocentesis. As such, 36 patients were excluded; 84 patients were included for analysis, and all 84 patients agreed to participate. The 2018 International Consensus Meeting Criteria (ICM 2018) was used for the classification of 49 patients with PJI (score ≥ 6) and 35 without PJI (score ≤ 2). The classification was used as the standard against which the different timings for reading LE strips were compared. All patients without PJI were followed for more than 1 year, during which they did not report the occurrence of PJI. All patients were graded against the diagnostic criteria regardless of their LE strip test results. In 83 patients, one drop of synovial fluid (50 µL) was applied to LE strips before and after centrifugation, and in one patient (without PJI), the sample was not centrifuged because the sample volume was less than 1.5 mL. The results of the strip test were read on an automated colorimeter. Starting from 1 minute after centrifugation, these strips were automatically read once every minute, 15 times (over a period of 16 minutes), and the results were independently recorded by two observers. Results were rated as negative, ±, 1+, and 2+ upon the machine reading. Grade 2+ (dark purple) was used as the threshold for a positive result. An investigator who was blinded to the study performed the statistics. Optimal timing for reading the LE strip before and after centrifugation was determined by using receiver operative characteristic (ROC) analysis. The specificity, sensitivity, and positive predictive and negative predictive values were calculated for key timepoints. RESULTS: Before centrifugation, the area under the curve was the highest when the results were read at 5 minutes (0.90 [95% CI 0.83 to 0.98]; sensitivity 0.88 [95% CI 0.75 to 0.95]; specificity 0.89 [95% CI 0.72 to 0.96]). After centrifugation, the area under the curve was the highest when the results were read at 10 minutes (0.92 [95% CI 0.86 to 0.98]; sensitivity 0.65 [95% CI 0.50 to 0.78]; specificity 0.97 [95% CI 0.83 to 1.00]). CONCLUSION: The LE strip test results are affected by time and centrifugation. For samples without centrifugation, we found that 5 minutes after application was the best time to read LE strips. We cannot deny the use of centrifuges because this is an effective way to solve the sample-mingling problem at present. We recommend 10 minutes postapplication as the most appropriate time to read LE strips after centrifugation. Multicenter and large-sample size studies are warranted to further verify our conclusion. LEVEL OF EVIDENCE: Level II, diagnostic study.

Global transcriptome dissection of pollen-pistil interactions induced self-incompatibility in dragon fruit (Selenicereus spp.).

Self-incompatibility (SI) is a major issue in dragon fruit (Selenicereus spp.) breeding and production. Therefore, a better understanding of the dragon fruit SI mechanism is needed to improve breeding efficiency and ultimate production costs. To reveal the underlying mechanisms of SI in dragon fruit, plant anatomy, de novo RNA sequencing-based transcriptomic analysis, and multiple bioinformatic approaches were used to analyze gene expression in the pistils of the self-pollinated and cross-pollinated dragon fruit flowers at different intervals of time after pollination. Using fluorescence microscopy, we observed that the pollen of 'Hongshuijing', a self-incompatible dragon fruit variety (S. monacanthus), germinated on its own stigma. However, the pollen tube elongation has ceased at 1/2 of the style, confirming that dragon fruit experiences gametophyte self-incompatibility (GSI). We found that the pollen tube elongation in vitro was inhibited by self-style glycoproteins in the SI variety, indicating that glycoproteins were involved in SI. That is to say the female S factor should be homologous of S-RNase or PrsS (P. rhoeas stigma S factor), both of which are glycoproteins and are the female S factors of the two known GSI mechanism respectively. Bioinformatics analyses indicated that among the 43,954 assembled unigenes from pistil, there were six S-RNase genes, while 158 F-box genes were identified from a pollen transcriptomic dataset. There were no P. rhoeas type S genes discovered. Thus, the identified S-RNase and F-box represent the candidate female and male S genes, respectively. Analysis of differentially expressed genes (DEGs) between the self and cross-pollinated pistils at different time intervals led to the identification of 6,353 genes. We then used a weighted gene co-expression network analysis (WGCNA) to find some non-S locus genes in SI responses in dragon fruit. Additionally, 13 transcription factors (TFs) (YABBY4, ANL2, ERF43, ARF2, BLH7, KNAT6, PIF3, two OBF1, two HY5 and two LHY/CCA) were identified to be involved in dragon fruit GSI. Thus, we uncovered candidate S and non-S genes and predicted more SI-related genes for a more detailed investigation of the molecular mechanism of dragon fruit SI. Our findings suggest that dragon fruit possesses a GSI system and involves some unique regulators. This study lays the groundwork for future research into SI mechanisms in dragon fruit and other plant species.

Metabolomics and Transcriptomics Analysis of Pollen Germination Response to Low-Temperature in Pitaya (Hylocereus polyrhizus).

Cross-pollination can improve the percentage of fruit set and fruit weight for most red flesh varieties in pitaya. The technology of pollen storage was very important for successful cross-pollination. However, till present, the technology of pollen storage is unsatisfactory in pitaya production. In this study, pitaya pollen stored at low temperature was taken as the research object, and its physicochemical indexes, metabolomics, and transcriptomics were studied. The results showed that in vitro pollen germination rate decreased significantly with the increase in storage time. Soluble sugar and soluble protein content of pollen peaked on the first day of storage, whereas its relative conductivity, and manlondialdehyde (MDA) and proline contents increased gradually during storage. At the same time, the antioxidant enzyme system of pollen was also affected. Superoxide dismutase (SOD) activity decreased, while the activities of catalase (CAT) and peroxidase (POD) increased and superoxide anion generation rate increased gradually during storage. According to the metabolomics results, amino acid, peptide, nucleotide, plant hormone, terpene, alcohol, phenol, flavonoid, sterol, vitamin, ester, sphingolipid, and ketone contents increased significantly during storage, whereas flavonoid and pigment contents declined gradually. During pollen storage, the gene expressions related to carbohydrate metabolism, protein metabolism, acid and lipid metabolism, sterol metabolism, plant hormone metabolism, and signal transductions were significantly downregulated. With KEGG pathway analysis, isoquinoline alkaloid biosynthesis, tyrosine metabolism, alanine, aspartate, and glutamate metabolism of pollen were affected significantly during low-temperature storage. Correlation analysis showed that the gene expression patterns of HuRP2, HuUPL1, and HuAAT2 had significant effects on pollen germination. D-arabinose 5-phosphate and myricetin were positively correlated with pollen germination rate, which was valuable for studying preservation agents. In this study, the changes in pollen during low-temperature storage were described from the level of metabolites and genes, which could provide theoretical support for the research and development of pollen long-term storage technology in pitaya.

Posteromedial Corner Release with the Knee in Figure-of-Four Position vs Conventional Position for Varus Knee Arthroplasty.

OBJECTIVE: To introduce posteromedial corner release with the knee in the figure-of-four position versus the conventional position for varus knee arthroplasty. METHODS: This is a retrospective study. From March 2015 to September 2019, a series of 123 patients (139 knees) with varus knee were randomly and blindly allocated to experimental group (60 patients; 68 knees) and control group (57 patients; 65 knees). Patients in experimental group underwent posteromedial corner release with the knee in the figure-of-four position; and patients in control group with the knee in the conventional position. If soft tissue balance was not completely achieved or the medial gap was still tight, an additional loosening technique were used to achieve symmetric medial and lateral space in both groups. Time for soft tissue balancing was defined as the time from the start of the spacer test to the end of the balance test. Length of release was defined as the distance from the osteotomy surface of the tibial plateau to the farthest structures released. The rating system of Hospital for Special Surgery (HSS) knee score was used to evaluate the clinical results. Quantitative variables were described as mean and standard deviation, and compared by one-way analysis of variance. RESULTS: The mean age of experimental group and control group was 70.2 ± 8.7 years and 68.7 ± 6.2 years, respectively (P > 0.05). Preoperatively, the mean HSS score of the groups was 38.2 ± 11.3 and 39.1 ± 10.7, respectively (P > 0.05). The mean varus knee angle was 19.7° ± 9.3° and 19.3° ± 10.7°, respectively (P > 0.05). The mean time for soft tissue balancing was 8.4 ± 3.3 min and 11.3 ± 6.9 min in experimental and control group, respectively (P < 0.05). The mean length of releasing posteromedial corner structures was 35.5 ± 13.4 mm and 27.3 ± 9.7 mm in experimental and control group, respectively (P < 0.05). Additional special loosening techniques were performed in eight knees in experimental group and seven knees in control group. The HSS scores 5 years after surgery were 95.1 ± 16.9 and 94.8 ± 17.2 respectively (P > 0.05). No complications were found during the follow-up time, and the clinical symptoms were observed to be significantly improved in the patients. CONCLUSION: The posteromedial corner can be released more extensively and thoroughly when the knee is placed in the figure-of-four position during varus knee arthroplasty.

The complete genome of Banana streak GF virus Yunnan isolate infecting Cavendish Musa AAA group in China.

Banana streak virus (BSV) belongs to the members of the genus Badnavirus, family Caulimoviridae. At present, BSV contains nine species in the International Committee on Taxonomy of Viruses (ICTV) classification report (2018b release). Previous study indicated that the viral particles of Banana streak virus Acuminata Yunnan (BSV-Acum) were purified from banana (Cavendish Musa AAA group) leaves in Yunnan Province, China, and its complete genome was obtained. To further determine whether this sample infecting with Banana streak GF virus (BSGFV), the polymerase chain reaction (PCR) cloning and complete genome analysis of the Banana streak GF virus Yunnan isolate (BSGFV-YN) isolate were carried out in this study. The result showed that BSGFV-YN infecting Cavendish Musa AAA group was co-infecting this sample. Its genome contains a total of 7,325 bp in length with 42% GC content. This complete genome sequence was deposited in GenBank under accession number MN296502. Sequence analysis showed that the complete genome of BSGFV-YN was 98.14% sequence similarity to BSGFV Goldfinger, while it was 49.10-57.09% to other BSV species. Two phylogenetic trees based on the complete genome and ORFIII polyprotein indicated that BSGFV-YN and other BSV species clustered into a group, while it was the highest homology with BSGFV Goldfinger. Although BSGFV-YN and BSGFV Goldfinger were highly homologous, their cultivating bananas are different. The former cultivating banana was from Cavendish Musa AAA group, while the latter cultivating banana was from Goldfinger Musa AAAB group. Compared with BSGFV Goldfinger, the genome of BSGFV-YN has an extra multiple repetitive sequences in the intergenetic region between ORFIII and ORFI, suggesting that this region might be related to host selection. In summary, a BSGFV-YN distant from BSV-Acum was identified from the same sample, and its complete genome sequence was determined and analyzed. The study extends the polymorphism of BSVs in China and provides scientific clue for the evolutionary relationship with host selection of badnaviruses.

Ethyl acetate fraction of flavonoids from Polygonum hydropiper L. modulates pseudorabies virus-induced inflammation in RAW264.7 cells via the nuclear factor-kappa B and mitogen-activated protein kinase pathways.

Pseudorabies virus (PRV) infection leads to severe inflammatory responses and tissue damage, and many natural herbs exhibit protective effects against viral infection by modulating the inflammatory response. An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. (FEA) was prepared through ethanol extraction and ethyl acetate fractional extraction. An inflammatory model was established in RAW264.7 cells with PRV infection to evaluate the anti-inflammatory activity of FEA by measuring cell viability, nitric oxide (NO) production, reactive oxygen species (ROS) release, and mRNA expression of inflammatory factors, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Its functional mechanism was investigated by analyzing the phosphorylation and nuclear translocation of key proteins in the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Our findings indicate that PRV induced inflammatory responses in RAW264.7 cells, and the responses were similar to that in lipopolysaccharide (LPS)-stimulated cells. FEA significantly suppressed NO synthesis and down-regulated both expression and secretion of COX-2, iNOS, and inflammatory cytokines (P<0.05 or P<0.01). FEA also reduced NF-κB p65 translocation into the nucleus and decreased MAPK phosphorylation, indicating that the NF-κB/MAPK signaling pathway may be closely related to the inflammatory response during viral infection. The findings suggested the potential pharmaceutical application of FEA as a natural product that can treat viral infections due to its ability to mitigate inflammatory responses.

Modern instant messaging platform for postoperative follow-up of patients after total joint arthroplasty may reduce re-admission rate.

BACKGROUND: Follow-up after artificial joint replacement greatly helps achieve surgical outcomes. Mobile internet technology and mobile terminal equipment may increase the effectiveness of artificial joint replacement. However, only a few studies have evaluated the effectiveness of this technology. We aimed to analyze the reasons and outcomes of patients who used the instant messaging platform after undergoing artificial joint replacement. METHODS: Among the 548 cases of arthroplasty (250 hips, 298 knees) performed between December 2015 and June 2018 in the Department of Joint Surgery of our institution; 358 (164 hip joints, 194 knee joints) participated in instant messaging platform consultation, whereas the remaining 190 (86 hip joints, 104 knee joints) participated in traditional telephone consultation, as a control group. Follow-up time was from December 2015 to August 2018 (follow-up period was 2-32 months). Data on age, sex, type of surgery, date of surgery, date of discharge, and length of hospital stay were collected from electronic medical records. RESULTS: We analyzed the consultation contents of 358 patients who participated in instant messaging platform consultation. Counseling was mainly related to pain (13.6%), appointment review (12.4%), activity problems (10.5%), and incision problems (8.9%). Most problems were resolved through online guidance, with 8.4% of patients requiring only outpatient treatment and 2.5% of patients requiring rehospitalization. A total of 190 patients were followed up through traditional telephone consultation; 6.8% of patients required outpatient department treatment and 7.4% were eventually re-admitted. CONCLUSION: The instant messaging platform consultation service effectively informs patients of potential postoperative problems and helps resolve them. It allows early detection and management of postoperative adverse events, including problems related to medication, wound, and activity, thereby effectively reducing readmission rate.

SSGA and MSGA: two seed-growing algorithms for constructing collaborative subnetworks.

The establishment of a collaborative network of transcription factors (TFs) followed by decomposition and then construction of subnetworks is an effective way to obtain sets of collaborative TFs; each set controls a biological process or a complex trait. We previously developed eight gene association methods for genome-wide coexpression analysis between each TF and all other genomic genes and then constructing collaborative networks of TFs but only one algorithm, called Triple-Link Algorithm, for building collaborative subnetworks. In this study, we developed two more algorithms, Single Seed-Growing Algorithm (SSGA) and Multi-Seed Growing Algorithm (MSGA), for building collaborative subnetworks of TFs by identifying the fully-linked triple-node seeds from a decomposed collaborative network and then growing them into subnetworks with two different strategies. The subnetworks built from the three algorithms described above were comparatively appraised in terms of both functional cohesion and intra-subnetwork association strengths versus inter-subnetwork association strengths. We concluded that SSGA and MSGA, which performed more systemic comparisons and analyses of edge weights and network connectivity during subnetwork construction processes, yielded more functional and cohesive subnetworks than Triple-Link Algorithm. Together, these three algorithms provide alternate approaches for acquiring subnetworks of collaborative TFs. We also presented a framework to outline how to use these three algorithms to obtain collaborative TF sets governing biological processes or complex traits.

Corrigendum: Hydrogen sulfide-induced itch requires activation of Cav3.2 T-type calcium channel in mice.

This corrects the article DOI: 10.1038/srep16768.

Phylogeographic pattern suggests a general northeastward dispersal in the distribution of Machilus pauhoi in South China.

Machilus pauhoi Kanehira is an important timber species in China. A provenance trial was recently set up to evaluate the growth performance of trees from different localities, with the aim of designing seed transfer guidelines. Here, we tested twelve nuclear microsatellite markers derived from other species of the Lauraceae family and investigated population genetic structure in M. pauhoi. Both the number of observed alleles per locus (Na) and the polymorphic information content (PIC) significantly decreased against the latitude, but showed an insignificant decrease against the longitude. Heterozygosity (Ho) and gene diversity (h) exhibited a weak correlation with geographic location. Private alleles were present in multiple populations, and a moderate level of population genetic differentiation was detected (Gst = 0.1691). The joint pattern of genetic diversity (Na, PIC, Ho, and h) suggests that general northeastward dispersal led to the current distribution of M. pauhoi. Significant but weak effects of isolation-by-distance (IBD) occurred, implicating the mountain ranges as the major barrier to gene flow. Both STRUCTURE and hierarchical clustering analyses showed three distinct groups of populations related to the physical connectivity among mountain ranges. A priority in designing genetic conservation should be given to the populations at the southwest side of the species' distribution. This conservation strategy can also be combined with the pattern of adaptive genetic variation from the provenance trial for comprehensive genetic resource management of native M. pauhoi.

Peripheral and spinal 5-HT receptors participate in cholestatic itch and antinociception induced by bile duct ligation in rats.

Although 5-HT has been implicated in cholestatic itch and antinociception, two common phenomena in patients with cholestatic disease, the roles of 5-HT receptor subtypes are unclear. Herein, we investigated the roles of 5-HT receptors in itch and antinociception associated with cholestasis, which was induced by common bile duct ligation (BDL) in rats. 5-HT-induced enhanced scratching and antinociception to mechanical and heat stimuli were demonstrated in BDL rats. 5-HT level in the skin and spinal cord was significantly increased in BDL rats. Quantitative RT-PCR analysis showed 5-HT1B, 5-HT1D, 5-HT2A, 5-HT3A, 5-HT5B, 5-HT6, and 5-HT7 were up-regulated in peripheral nervous system and 5-HT1A, 5-HT1F, 5-HT2B, and 5-HT3A were down-regulated in the spinal cord of BDL rats. Intradermal 5-HT2, 5-HT3, and 5-HT7 receptor agonists induced scratching in BDL rats, whereas 5-HT3 agonist did not induce scratching in sham rats. 5-HT1A, 5-HT2, 5-HT3, and 5-HT7 agonists or antagonists suppressed itch in BDL rats. 5-HT1A agonist attenuated, but 5-HT1A antagonist enhanced antinociception in BDL rats. 5-HT2 and 5-HT3 agonists or antagonists attenuated antinociception in BDL rats. Our data suggested peripheral and central 5-HT system dynamically participated in itch and antinociception under cholestasis condition and targeting 5-HT receptors may be an effective treatment for cholestatic itch.

Screening Reliable Reference Genes for RT-qPCR Analysis of Gene Expression in Moringa oleifera.

Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera.

Hydrogen sulfide-induced itch requires activation of Cav3.2 T-type calcium channel in mice.

The contributions of gasotransmitters to itch sensation are largely unknown. In this study, we aimed to investigate the roles of hydrogen sulfide (H2S), a ubiquitous gasotransmitter, in itch signaling. We found that intradermal injection of H2S donors NaHS or Na2S, but not GYY4137 (a slow-releasing H2S donor), dose-dependently induced scratching behavior in a µ-opioid receptor-dependent and histamine-independent manner in mice. Interestingly, NaHS induced itch via unique mechanisms that involved capsaicin-insensitive A-fibers, but not TRPV1-expressing C-fibers that are traditionally considered for mediating itch, revealed by depletion of TRPV1-expressing C-fibers by systemic resiniferatoxin treatment. Moreover, local application of capsaizapine (TRPV1 blocker) or HC-030031 (TRPA1 blocker) had no effects on NaHS-evoked scratching. Strikingly, pharmacological blockade and silencing of Cav3.2 T-type calcium channel by mibefradil, ascorbic acid, zinc chloride or Cav3.2 siRNA dramatically decreased NaHS-evoked scratching. NaHS induced robust alloknesis (touch-evoked itch), which was inhibited by T-type calcium channels blocker mibefradil. Compound 48/80-induced itch was enhanced by an endogenous precursor of H2S (L-cysteine) but attenuated by inhibitors of H2S-producing enzymes cystathionine γ-lyase and cystathionine ß-synthase. These results indicated that H2S, as a novel nonhistaminergic itch mediator, may activates Cav3.2 T-type calcium channel, probably located at A-fibers, to induce scratching and alloknesis in mice.

[Changes of tissue factor and tissue factor pathway inhibitor in neonatal jaundice due to infection].

OBJECTIVE: Tissue factor (TF) is an important factor in extrinsic coagulation. Tissue factor pathway inhibitor (TFPI) is a negative regulator of coagulation mediated by TF. Studies on TF and TFPI focus mainly on adult objects, seldom have been done on newborns, especially on sick newborns. The aim of this study was to observe the changes of TF and TFPI in plasma of newborns with infection jaundice and to research the effect of jaundice and infection on the balance of TF and TFPI in newborns. METHODS: The content of TF and TFPI in plasma of 21 jaundiced newborns with infection and 8 jaundiced newborns without infection as control was determined quantitatively with the enzyme-linked immunosorbent assay (ELISA). RESULTS: The content of TFPI and TF in plasma of jaundiced newborn with infection was significantly higher than that of controls [TFPI (21.0 +/- 4.3) vs. (16.2 +/- 1.9) microg/L, P < 0.01; TF (177 +/- 79) vs. (51 +/- 24) ng/L, P < 0.01]. The ratio of TFPI/TF was significantly lower in newborn with infection jaundice than the controls (137 +/- 61 vs. 319 +/- 67, P < 0.01). The 21 jaundiced newborns with infection were divided into the severe hyperbilirubinemia group (serum bilirubin > or = 205.2 micromol/L, n = 10) and the mild hyperbilirubinemia group (serum bilirubin < 205.2 micromol/L, n = 11). There was no significant difference of TFPI level between the severe hyperbilirubinemia group and mild hyperbilirubinemia group (P > 0.05). The TF content in the severe hyperbilirubinemia group was higher than that in the mild hyperbilirubinemia group (216 +/- 79 vs.141 +/- 63, P < 0.01), while the ration of TFPI/TF was lower in the severe hyperbilirubinemia group than in the mild hyperbilirubinemia group (100 +/- 30 vs. 171 +/- 74, P < 0.01). CONCLUSION: Infection might induce imbalance between the coagulation inhibition and activation in newborns. Hyperbilirubinemia can aggravate the imbalance induced by the infection through increasing plasma TF level.