Ultrasensitive quantitation of Paraquat based on a small molecule-induced dual-cycle amplification strategy.

Paraquat (PQ) is a typical biotoxic small molecule. Knowledge of how to directly introduce it into cyclic amplification rather than transform it into a secondary target is lacking in current analytical methods. Considering the urgent need for trace pesticide residue detection and the inherent defects of small molecule analysis, a CRISPR/Cas12a-driven small molecule-induced dual-cycle strategy was developed based on the immune competition method. The key to signal amplification is the mutual activation and acceleration between Cycle 1 triggered by the small molecule and Cycle 2 driven by CRISPR/Cas12a. Impressively, small molecules have been successfully incorporated into the dual-cycle strategy, which achieves a low detection limit (3.1 pg/mL) and a wide linear range (from 10 pg/mL to 50 µg/mL). Moreover, the designed biosensor was successfully employed to evaluate the PQ residual level in real samples and showed effective implementation for the bioanalysis of small molecule targets and pesticide residue-related food safety.

Continuous renal replacement therapy combined with double filtration plasmapheresis in the treatment of severe lupus complicated by serious bacterial infections in children: A case report.

There are few reports available on the combination therapy of continuous renal replacement therapy (CRRT) and double filtration plasmapheresis (DFPP) in patients with systemic lupus erythematosus (SLE) complicated by severe bacterial infections, especially in children. A 14-year-old female child with recurrent SLE complicated by severe sepsis-induced multiple organ dysfunction syndrome was administered CRRT combined with DFPP for blood purification in addition to routine immunosuppressant therapy. The changes in autoantibodies, cytokines, and coagulation function indexes of the patient before and after treatment were compared to explore the effect of such therapy on progression and prognosis. After DFPP therapy, significant decreases in the levels of double-stranded DNA antibody, cytokines interleukin (IL)-6, IL-10, and procalcitonin (PCT) were observed. Fibrinogen (Fib) decreased and needed to be replenished following DFPP. After CRRT combined with DFPP, the patient began to urinate sparingly (urine volume was < 50 mL/day) at the seventh week, the urine volume was > 400 mL/day (up to 560 mL/day) at the ninth week (63 days), and the urine volume was >1,000 mL/day at the tenth week, at which time the renal function had fully recovered. DFPP may reduce the plasma Fib concentration, which needs to be replenished in a timely manner. CRRT combined with DFPP shows efficacy in patients with SLE, but the coagulation function requires close monitoring.

[Prognostic Factors Analysis of Children with Hemophagocytic Lymphohistiocytosis].

OBJECTIVE: To analyze the risk factors affecting prognosis of children with hemophagocytic lymphohistiocytosis (HLH). METHODS: The clinical manifestations and laboratory data of 143 HLH children who met the HLH-2004 diagnostic criteria in Shenzhen Children's Hospital from January 2009 to May 2017 were retrospectively analyzed, and the independent factors affecting prognosis were also analyzed. RESULTS: The median age of 143 HLH children was 1.9 (0.1-14.3) years old, and the median follow-up time was 6.7 years (1 day - 11.9 years). The overall survival rate of 1 month, 1 year, and 10 years was (87.4±5.5)%, (81.1±6.5)%, and (81.1±6.5)%, respectively. The deaths occurred within 1 year after onset. Multivariate analysis showed that central nervous system (CNS) involvement (P=0.047), low hemoglobin (P=0.002), prolonged activated partial thromboplastin time (APTT) (P<0.001), high triglyceride (P=0.005) were all the independent risk factors affecting survival of the children. Receiver operating characteristic curve indicated that APTT (AUC=0.753, P<0.001) was more valuable than other risk factors in predicting death of the children. The cut-off value of APTT was 56.6 s, and the sensitivity and specificity of which was 55.6% and 89.7%, respectively. CONCLUSION: Hypohemoglobinemia, prolonged APTT, hypertriglyceridemia, and CNS involvement the risk factors affecting prognosis of HLH, and prolonged APTT shows a strong predictive value for death.

Use of a modified tracheal tube in a child with traumatic bronchial rupture: A case report and review of literature.

BACKGROUND: Trauma is one of the leading causes of death in the pediatric population. Bronchial rupture is rare, but there are potentially severe complications. Establishing and maintaining a patent airway is the key issue in patients with bronchial rupture. Here we describe an innovative method for maintaining a patent airway. CASE SUMMARY: A 3-year-old boy fell from the seventh floor. Oxygenation worsened rapidly with pulse oxygen saturation decreasing below 60%, as his heart rate dropped. Persistent pneumothorax was observed with insertion of the chest tube. Fiberoptic bronchoscopy was performed, which confirmed the diagnosis of bronchial rupture. A modified tracheal tube was inserted under the guidance of a fiberoptic bronchoscope. Pulse oxygen saturation improved from 60% to 90%. Twelve days after admission, right upper lobectomy was performed using bronchial stump suture by video-assisted thoracic surgery without complications. A follow-up chest radiograph showed good recovery. The child was discharged from hospital three months after admission. CONCLUSION: A modified tracheal tube could be selected to ensure a patent airway and adequate ventilation in patients with bronchial rupture.

[Clinical features of children with influenza and plastic bronchitis: an analysis of 63 cases].

OBJECTIVE: To study the clinical features of children with influenza and plastic bronchitis (PB). METHODS: A retrospective analysis was performed on the medical data of 63 children with influenza and PB, including clinical manifestations, laboratory examination results, imaging findings, treatment, and outcome. RESULTS: Among the 63 children, there were 52 boys (83%) and 11 girls (17%), and 42 children had influenza A and 21 had influenza B. Among these children, 38 (60%) aged 3-6 years, and 15 (24%) had underlying diseases. The main clinical manifestations were high fever (90%), cough (95%), and shortness of breath (73%). Twenty-four children (38%) were found to have atelectasis by imaging examination. Auscultation showed that 16 children (25%) had no rales in the lungs. Of all children, 41 were admitted to the intensive care unit, and 32 required mechanical ventilation. All children underwent fiberoptic bronchoscopy and alveolar lavage. Among the 63 children, 60 recovered and 3 died. CONCLUSIONS: Influenza with PB is often observed in boys and preschool children. For influenza children with shortness of breath, even if there is no atelectasis on chest X-ray or no rales are found by auscultation, the possibility of PB still needs to be considered.

[Clinical application of cardiac output monitoring in children with severe hand-foot-mouth disease].

OBJECTIVE: Significant cardiac dysfunction has been found in children with severe hand-foot-mouth disease and heart failure is the major cause of death in these patients. Evaluation of cardiac function is essential for the treatment of severe cases. This study evaluated the clinical value of cardiac output monitoring in children with severe hand-foot-mouth disease. METHODS: A total of 107 children with severe hand-foot-mouth disease admitted to the pediatric intensive care unit from April 2011 to September 2011 were enrolled and divided into three groups by clinical stage: 73 cases in stage 2, 23 cases in stage 3 and 11 cases in stage 4. Cardiac output and stroke volume were measured by ultrasonic cardiac output monitors (USCOM). Ninety-five children received MRI scanning and were grouped according to the results of MRI: 41 cases (medulla oblongata involvements in 9 cases) in abnormal MRI group and 54 cases in normal MRI group. Cardiac output was compared between the children in different clinical stages and between different MRI results. RESULTS: Compared with children in clinical stages 2 and 3, cardiac output in children in clinical stage 4 decreased significantly (P<0.05). There was no differences in cardiac output between the normal and abnormal MRI groups, however cardiac output was significantly lower in children with medulla oblongata involvement than in those with other involvements and normal MRI. CONCLUSIONS: Significant decrease in cardiac output suggests critical conditions and medulla oblongata cardiovascular center involvement in children with severe hand-foot-mouth disease. Dynamic measurement of cardiac output is valuable for treatment of the disease.

[Characteristics of severely and critically ill children with 2009 influenza A (H1N1) virus infection].

OBJECTIVE: To analyze the clinical characteristics of severely and critically ill children with 2009 influenza A (H1N1) infection. METHOD: Clinical data of 150 cases with 2009 influenza A (H1N1) virus infection confirmed with the use of a real-time polymerase-chain-reaction assay on nasopharyngeal swab specimens were analyzed. RESULT: Among 150 severely and critically ill children with 2009 influenza A (H1N1) virus infection, 103 were male, 47 were female; the median age was 5 years, 81(55%) were 5 years of age or older; 21 (14%) had underlying chronic diseases. The most common presenting symptoms were fever (95%), cough (89%), vomiting (23%), wheezing (19%), abdominal pain (16%), lethargy (7%), seizures (6%), myalgia (6%), and diarrhea (6%). The common laboratory abnormalities were increased or decreased white blood cells counts (40%), elevated of CRP (33%), LDH (29%), CK (25%) and AST (19%). Clinical complications included pneumonia (65%), encephalopathy (12%), myocarditis (5%), encephalitis (1%) and myositis (1%). All patients had received antibiotics before admission or on admission; 73% of patients had received oseltamivir treatment, 23% of patients had received corticosteroids; 32 (21%) were admitted to an ICU, 13 patients were intubated and mechanically ventilated. Fourteen patients with dyspnea who were irresponsive to the treatment experienced bronchoalveolar lavage with flexible bronchoscopy, and the branching bronchial casts were removed in 5 patients. Totally 145 (97%) patients were discharged, five (3%) died, three previously healthy patients died from severe encephalopathy, one patient died from ARDS, one previously healthy patient died from secondary fungal meningitis. CONCLUSION: Severely and critically ill children with 2009 influenza A (H1N1) virus infection may occur mainly in older children without underlying chronic disease. The clinical spectrum and laboratory abnormality of the patients can have a wide range. Neurologic complications may be common and severe encephalopathy can lead to death in previously healthy children. Early use of bronchoalveolar lavage with flexible bronchoscopy may reduce death associated with pulmonary complications.

[Changes of immune function in patients with 2009 influenza A (H1N1)].

OBJECTIVE: To investigate the alteration of immune function and possible immunopathogenesis in the children with 2009 influenza A (H1N1) infection. METHOD: Sixty patients with 2009 influenza A (H1N1) infection hospitalized in Shenzhen Children's Hospital between November 1, 2009 and January 10, 2010 and 20 age-matched healthy children were enrolled in this study. The patients were divided into two groups according to the severity of influenza A infection: 35 mild cases (mild pneumonia) and 25 severe cases (severe pneumonia, acute encephalopathy associated with influenza A, and 3 died from acute necrotizing encephalopathy with influenza A infection). Real-time PCR was used to evaluate the expression levels of pattern recognition receptor (PRRs), retinoic acid induced gene I/melanoma differentiation associated gene 5 (RIG/MDA5), Toll-like receptors (TLRs) and TLRs signaling molecules, and negative-regulator. Three color fluorescent and flow cytometry were used to investigate the apoptosis of CD3(+), CD4(+), CD8(+) and CD19(+) cells. Plasma cytokines (IL-1ß, IL-6, TNF-α, IFN-γ, IFN-α, IL-10) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). RESULT: (1) The expression levels of RIG/MDA5, TLR2, 4 were much higher in the patients with influenza A infection, especially severe cases [TLR2 (9.69 ± 3.15) × 10(-2) vs. (3.96 ± 0.83) × 10(-2), t = 10.16, P < 0.05; TLR4 (10.23 ± 2.85) × 10(-2) vs. (7.46 ± 2.18) × 10(-2), t = 3.76, P < 0.05]. The expression levels of TLRs signal transduction molecules like MyD88 and TRAM also increased. (2) The cell counts of CD3(+), CD4(+), CD8(+) T cells and NK cells were markedly lower in the patients with influenza A infection compared to the NC group [CD3(+)(1.22 ± 0.38) × 10(9)/L vs.(3.59 ± 1.10) × 10(9)/L, t = 9.21, P < 0.05]. (3) Plasma concentrations and the mRNA expression of TNF-α, IL-6, and IL-1ß were elevated in mild cases, while declined in severe cases [TNF-α (6.42 ± 1.76) × 10(-2) vs. (9.05 ± 2.51) × 10(-2), t = 4.55, P < 0.05]. Plasma concentrations of IFN-α/IFN-ß were up-regulated gradually with the aggravation of the disease, especially in severe cases. Compared with healthy controls, the expression of IFN-I inducible gene IP-10, RANTES, or iNOS was significantly higher in children with mild [IP-10 (20.52 ± 6.09) × 10(-2) vs.(1.18 ± 0.34) × 10(-2), t = 18.74, P < 0.05], and relatively lower in severe cases. (4) The apoptosis of CD3(+), CD4(+), CD8(+) and NK cells significantly increased in the patients with influenza A infection than those in NC group [CD3(+)(32.90 ± 7.66)% vs. (20.21 ± 6.58)%, t = 6.21, P < 0.05]. Compared with healthy controls, the expression levels of apoptosis-related gene like TRAIL and CASPASE-3 significantly increased in the patients with influenza A infection. (5) The expression levels of negative regulator of SOCS1, SOCS3, IRAK-M, TRAF4 and FLN29 were significantly increased in the patients with influenza A, especially in severe cases than those in NC group (P < 0.05). CONCLUSION: Immune function changed with the severity of the disease. The mild cases presented systemic immune activation status, while critically ill cases presented mixed immune activation and immunosuppression status.

[Role of transduce molecules and modulatory factors of signal pathways of Toll-like receptor in inflammatory response of children with sepsis].

OBJECTIVE: To investigate the role of transduce molecules and modulatory factors of signal pathways of Toll-like receptors (TLRs) in aberrant inflammatory response in children with sepsis. METHODS: Peripheral blood mononuclear cell (PBMC) and serum were obtained from 10 children with sepsis, 13 children with severe sepsis and 17 age-matched healthy children as controls. Real-time polymerase chain reaction (real-time PCR) were used to evaluate the mRNA expression levels of TLR2, TLR4, myeloid differentiation primary response gene 88(MyD88), interleukin-1 receptor-associated kinase 4 (IRAK-4), tumor necrosis factor receptor-associated factor 6 (TRAF6), TAK1-binding protein 2 (TAB2) transforming growth factor-beta-activating kinase 1 (TAK1), interleukin-1 receptor-associated kinase 3 (IRAK-M), zinc finger protein inhibiting nuclear factor-KappaB (Triad3A), protein associated with Tlr4 (PRAT4B) and signal-transducing adaptor protein-2 (STAP2), and the levels of mRNA expression and production of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and IL-6] were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the healthy control group, the levels of gene expression and protein of pro-inflammatory cytokines (TNF-alpha, IL-1 beta and IL-6), the levels of gene expression of signal pathway molecules of TLRs (TLR2, TLR4, MyD88, TRAF6, IRAK4, TAK1 and TAB2) and the levels of gene expression of positive modulators of TLRs pathways (PRAT4B and STAP2) were significantly increased in sepsis group and severe sepsis group (all P < 0.01), and the levels in severe sepsis group were even higher than those in sepsis group (all P < 0.01). The mRNA expression levels of negative modulators of TLRs pathways (IRAK-M and Triad3A) were significantly elevated in sepsis group compared with those in healthy control group (all P < 0.01), but the mRNA expression levels of the negative modulators in severe sepsis group were significantly lowered compared with those in sepsis group (all P < 0.01). CONCLUSION: Aberrant expression of signal pathway molecules and the modulators in TLRs signaling might play an important role on production and development of abnormal inflammatory response in children with sepsis.

[Randomized controlled study on acupuncture for treatment of diabetic paralytic squint].

OBJECTIVE: To observe clinical therapeutic effect of acupuncture on diabetic paralytic squint. METHODS: Seventy-two cases of diabetic paralytic squint were randomly divided into a medication group, an acupuncture group and an acupuncture and medication group. The medication group were treated with intramuscular injection of Methyl vitamin B12 250 microg, once daily; the acupuncture group were treated by acupuncture at different acupoints according to different paralytic muscles of eyes with adjuvant acupoints selected according to symptoms; the acupuncture and medication group were treated with the routine medicine and acupuncture. The treatment was given for 28 days. RESULTS: The total effective rate of 87. 5% in the acupuncture group and 95.7% in the acupuncture and medication group were higher than 54.5% in the medication group (P < 0.05, P < 0.01), with no significant difference between the acupuncture group and the acupuncture and medication group (P > 0.05). CONCLUSION: Acupuncture has a definite therapeutic effect on diabetic paralytic squint, which is better than that of routine medication.

[Changes and significance for regulatory factors for signal pathways of Toll-like receptors in immunological pathogenesis of Kawasaki disease].

OBJECTIVE: Many clinical evidences and epidemiologic data in the past suggested that Kawasaki disease (KD) is correlated with an acute immune dysfunction caused by infection. In our preliminary study, Toll-like receptor 4 signal pathway, which could activate nuclear transcription factor-kappaB and induce excessive product of proinflammatory cytokines, chemokines and co-stimulatory molecules, was observed to be significantly activated during acute phase of Kawasaki disease. But the causative factors and regulatory mechanism are still unknown. In this study, the authors further investigated the changes and significances of regulatory factors for signal pathway of Toll-like receptors (TLRs) in immunological pathogenesis of Kawasaki disease. METHODS: Forty-eight children with KD, sixteen children with infectious disease (ID) and sixteen age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the expression levels of regulatory and effective factors in toll-like receptor 4 (TLR4) signal pathways and proinflammatory factors in peripheral blood monocyte/macrophage (MC). The expression of TLR4 protein in MC was analyzed by flow cytometry. RESULTS: (1) Expression levels of TLR4, MD-2, MyD88, IRAK-4, TRAF6, TAK1, TAB1 and TAB2 mRNA in KD group were elevated significantly during acute phase (P < 0.05). (2) Transcription levels of regulatory factors PRAT4B and STAP2 in patients with KD or ID were found to be higher than those in the healthy volunteers (P < 0.05), but no significant differences in these parameters were detected between KD patients and ID patients (P > 0.05). Transcription levels of regulatory factors such as FLN29, RP105 and MD-1 were up-regulated to some extents and expression level of DAP12 mRNA in KD patients were found to be lower than that in normal controls (P < 0.05), while all of the four regulatory factors were found to be lower than those in ID patients (P < 0.05). Expressions of proinflammatory cytokines such as L-1beta, IL-6 and TNF-alpha in KD patients were significantly higher than those in ID patients (P < 0.05). (3) Stimulation with lipopolysaccharide (LPS) elevated remarkably the expressions of regulatory factors PRAT4B and STAP2 in KD patients or healthy volunteers (P < 0.05). All of the four negative-regulatory factors were found to be significantly up-regulated after stimulation with LPS in controls (P < 0.05). No responses to LPS were observed in expression of FLN29, RP105 and MD-1 mRNA in KD patients (P > 0.05), except for increased transcription of DAP12. (4) The levels of PRAT4B and STAP2 mRNA in KD patients with coronary artery lesion (KD-CAL(+)) were detected to be higher than those in KD patients without coronary artery lesion (KD-CAL(-)) during acute phase (P < 0.05), while those of FLN29, RP105 and MD-1 in KD-CAL(+) group were lower than that in the latter (P < 0.05). No significant difference in DAP12 mRNA expression level was detected between the two groups (P > 0.05). Expressions of proinflammatory cytokines and TLR4 protein on surface of CD14-positive cells in KD-CAL(+) group were found to be higher than those in KD-CAL(-) group [(11.9 +/- 2.4)% vs. (6.5 +/- 1.7)%, P < 0.05]. CONCLUSION: Disturbance of negative-regulatory factors may be one of the factors causing aberrant immunological function in KD.

[Pathomorphological observation of the hypertrophic scar induced by injury to conical structure in female red Duroc pig].

UNLABELLED: To further explore the relationship between hypertrophic scar and injury to conical structure of skin and the pathogenesis of hypertrophic scar, and to reproduce an optimal animal model of hypertrophic scar. METHODS: The back of two FRDP pigs were shaved, and a piece of normal skin was harvested for the observation of conical structure of skin. Skin wounds with depth of 0.38 mm, 0.76 mm, 1.14 mm and 1.52 mm, respectively, were created by gas-driven dermatome. Eight wounds measuring 7.0 cm x 7.0 cm were created on each pig. The wounds were divided into 4 groups according to the wound depth with 4 wounds in each group, i.e. 0.38 mm group, 0.76 mm group, 1.14 mm group and 1.52 mm group. The 0.38 mm and 0.76 mm groups were designated as superficial wound groups and 1.14 mm and 1.52 mm groups as deep wound groups. The wounds were allowed to heal without treatment. Tissue samples from the wound were harvested on 0, 10, 30, 60, 90 and 150 post injury day (PID) , and they were sectioned for HE staining and staining for elastic fibers (VVG). The wound healing and the scar formation were observed with naked eye. The skin conical structures in normal and injured skin were also observed. The morphology of hypertrophic scar was observed, and the thickness of the scar tissue was determined and scored. RESULTS: The wounds in superficial wound groups healed within 3 weeks with flat surface without scar formation. The wounds in deep wound groups healed later than 4 weeks with thick, hairless, hard in texture, with depigmentation or pigmentation, finally forming contracture. The skin conical structure could be found on the back of FRDP with HE and VVG staining, and it was similar to that of human in terms of the structure. In superficial wound groups, the upper part of the skin conical structure was injured, but fat fornix and glands were intact. In deep wound groups, the lower part of the skin cone, together with the fat fornix and gland were all injured. On the 150th post injury day, the histological picture of the tissue in superficial wound groups was similar to that of normal skin. But the skin conical structure could not be found in deep wound groups, and the wounds were filled by a large accumulation of disarrayed and irregularly arranged collagen fibers. With passage of time, the scar became thicker and thicker, and the scar hypertrophy reached the zenith in 150th PID. CONCLUSION: The injury of skin conical structure can lead to the formation of hypertrophic scar. FRDP can be used to reproduce and ideal model of hypertrophic scar.