Impact of lead oxide nanoparticle exposure on the polarization of microglia cells in mouse hippocampus / 中国职业医学

Objective To investigate the effect of exposure to lead oxide nanoparticles (PbO NPs) on the polarization of microglia in mouse hippocampus. Methods i) Specific pathogen-free male C57 mice were randomly divided into control group, low-, medium- and high-dose groups, with 10 mice in each group. Mice in these three dose groups were intraperitoneally injected with PbO NPs suspension at doses of 5, 10 and 20 mg/kg per day, respectively, and mice in the control group were intraperitoneally injected with the same volume of 0.9% sodium chloride solution, five days per week for four weeks. ii) BV-2 cells were treated with PbO NPs at doses of 0.0, 2.5, 5.0 and 10.0 mg/L for 24 hours. iii) BV-2 cells were randomly divided into control group, PbO NPs group and triggering receptor expressed on myeloid cells 2 (TREM2) high expression + PbO NPs group. The cells in the control group received no treatment. The cells in PbO NPs group were exposed to 10.0 mg/L PbO NPs suspension for 24 hours. Cells in TREM2 high expression + PbO NPs group were transfected with Trem2 high expression plasmid, and then exposed to 10.0 mg/L PbO NPs suspension for 24 hours. iv) The mRNA expression of M1 markers ［nitric oxide synthase (iNos), cyclooxygenase 2 (Cox2), chemokine receptor 7 (Ccr7)］, M2 markers ［arginin-1 (Arg-1), transforming growth factor-β (Tgf-β), chemokine receptor 2 (Ccr2)］ and Trem2 of microglia was detected by real-time fluorescent quantitative polymerase chain reaction. The protein expression of iNOS, ARG-1 and TREM2 was detected by Western blotting. Results i) During the experiment, there was no significant difference in body weight of mice among these four groups (P>0.05). The relative expression of Cox2 and Ccr7 mRNA in the hippocampus of the mice increased in the low-dose group and the iNos, Cox2 and Ccr7 mRNA increased in the medium- and high-dose groups, compared with the control group (all P<0.05). The relative mRNA expression of Tgf-β in the hippocampus of the mice of low-dose group and Arg-1, Tgf-β and Ccr2 in the medium- and high-dose groups was decreased compared with the control group (all P<0.05). The mRNA relative expression of iNos, Cox2 and Ccr7 was increased (all P<0.05), while the mRNA relative expression of Arg-1, Tgf-β and Ccr2 was decreased (all P<0.05) in the hippocampus of the mice of high-dose group compared with the low-dose group. The relative expression of Trem2 mRNA and TREM2 protein in the hippocampus of mice of the medium- and high-dose groups was lower than those in the control group (all P<0.05). The relative expression of Trem2 mRNA and TREM2 protein in the hippocampus of mice of the high dose group was lower than those in the low- and the medium-dose groups (all P<0.05). With the increase of PbO NPs exposure dose, the relative expression of iNOS protein in hippocampus tissues of mice increased (P<0.01), and the relative expression of ARG-1 protein decreased (P<0.01). ii) With the increase of PbO NPs exposure dose, the relative expression of iNOS protein increased (P<0.01), and the relative expression of ARG-1 protein decreased (P<0.01) in BV-2 cells. The relative expression of iNOS protein in BV-2 cells of PbO NPs group and TREM2 high expression + PbO NPs group was increased (all P<0.05), and the relative expression of ARG-1 protein decreased (all P<0.05) compared with the control group. The relative expression of iNOS protein decreased (P<0.05), and the relative expression of ARG-1 protein increased (P<0.05) in BV-2 cells of TREM2 high expression + PbO NPs group compared with the PbO NPs group. Conclusion Exposure to PbO NPs could increase the M1 polarization and decrease the M2 polarization of microglia, with a dose-effect relationship. The M1 polarization of microglia decreased and M2 polarization increased after overexpression of Trem2 gene. The regulation of microglia polarization by TREM2 may be involved in the neurotoxic effects of PbO NPs.

Mechanism study of chlorogenic acid alleviating macrophage activation / 中国药房

OBJECTIVE To observe the effects of chlorogenic acid on the activation of macrophage induced by lipopolysaccharide （LPS）， and to explore the role of triggering receptors expressed on myeloid cells-2 （TREM2） in the action. METHODS To find a suitable LPS concentration， the cells were cultured with 1， 10 and 100 ng/mL LPS for 24 h. The level of interleukin 6 （IL-6） in the cell culture supernatant and protein expression of inducible nitric oxide synthase （iNOS） in the cells were detected. To search for a suitable chlorogenic acid concentration， the cells were divided into control group， LPS group and three chlorogenic acid （0.01， 0.1 and 1 μmol/L）+LPS groups. The levels of tumor necrosis factor α （TNF-α） and IL-1β in the cell culture supernatant， the protein expressions of iNOS and TREM2 in the cells and cell viability were detected. To observe the effects of TREM2 in chlorogenic acid alleviating macrophage activation， TREM2-small interfering RNA （TREM2-siRNA） was taken to intervene in TREM2 protein expression. The cells were divided into control group， LPS group， chlorogenic acid+LPS group， TREM2-siRNA+chlorogenic acid+LPS group and SC-siRNA+chlorogenic acid+LPS group. After 24 h incubation， the levels of TNF- α and IL-1β in the cell culture supernatant and protein expressions of TREM2， iNOS and nuclear factor κB p65 （NF-κB p65） in the cells were detected. RESULTS 10 ng/mL LPS promoted IL-6 release and increased iNOS protein expression， and 10 ng/mL LPS was taken in the next experiments. Compared with the LPS group， 0.1 μmol/L chlorogenic acid decreased TNF-α jiaji1981@126.com and IL-1β levels， and down-regulated iNOS expression，meanwhile increased TREM2 expression without effect on cell viability， and 0.1 μmol/L chlorogenic acid was taken in the next experiments. Compared with the control group， the protein expressions of iNOS and NF- κB p65 in the LPS group were significantly increased （P＜0.05）； compared with the LPS group， the protein expressions of iNOS and NF- κB p65 in the chlorogenic acid+LPS group were significantly decreased， the protein expressions of TREM2 was significantly increased （P＜ 0.05）； compared with the chlorogenic acid+LPS group， the protein expressions of iNOS and NF-κB p65 of TREM2-siRNA+ chlorogenic acid+LPS group were significantly increased， the protein expressions of TREM2 was significantly decreased （P＜0.05）. TREM2-siRNA could significantly reverse the above effects of chlorogenic acid， while SC-siRNA did not significantly affect the above anti-inflammatory effects of chlorogenic acid. CONCLUSIONS Chlorogenic acid can inhibit the LPS-induced macrophage activation， and its anti-inflammatory may be mediated by TREM2 protein.

Eventos paroxísticos que se confunden con epilepsia / Paroxysmal events that are confused with epilepsy

Resumen Los eventos paroxísticos no epilépticos (EPNE) se definen como episodios de aparición brusca y de breve duración que imitan a una crisis epiléptica, originados por una disfunción cerebral de origen diverso y a diferencia de la epilepsia no obedecen a una descarga neuronal excesiva. Su incidencia es mucho más elevada que la epilepsia y pueden aparecer a cualquier edad, pero son más frecuentes en los primeros años de vida. La inmadurez del sistema nervioso central en la infancia favorece que en este período las manifestaciones clínicas sean muy floridas y diferentes de otras edades. Fenómenos normales y comunes en el niño pueden también confundirse con crisis epilépticas. El primer paso para un diagnóstico correcto es establecer si este primer episodio corresponde a una crisis epiléptica o puede tratarse de un primer episodio de EPNE. Es importante seguir un protocolo de diagnóstico, valorando los antecedentes personales y familiares, sin olvidar el examen físico, analizar los posibles factores desencadenantes, los pormenores de cada episodio, si es posible un registro de los episodios, aplicar el sentido común y la experiencia y solamente proceder a los exámenes complementarios básicos como el registro EEG u otras exploraciones en caso de duda o para con firmación diagnóstica. En algunos casos se ha demostrado una base genética. Las opciones terapéuticas son escasas y la mayoría de EPNE tienen una evolución favorable.

Abstract Non-epileptic paroxysmal events (NEPE) are defined as episodes of sudden onset and short duration that mimic an epileptic seizure, caused by a brain dysfunction of diverse origin and, unlike epilepsy, are not due to excessive neuronal discharge. Its incidence is much higher than epilepsy and can appear at any age, but are more frequent in the first years of life. The immaturity of the central nervous system in childhood favors that in this period the clinical manifestations are more spectacular and different from other ages. Normal and common phenomena in children can also be confused with epileptic seizures. The first step for a correct diagnosis is to establish whether this first episode corresponds to an epileptic seizure or could be a first episode of NEPE. It is important to follow a diagnostic protocol, assessing the personal and family history, without forgetting the physical examination, analyzing the possible triggering factors, the details of each episode, if it's possible a record of the episodes, applying common sense and experience and only carrying out basic complementary tests such as EEG recording or others in case of doubt or for diagnostic confirmation. In some cases, a genetic basis has been demonstrated. Therapeutic op tions are scarce and the majority of NEPE have a favorable evolution.

Relationship between CCL21 and TREM2/DAP12 signaling pathways in spinal dorsal horn in remifentanil-induced hyperalgesia in mice with incisional pain / 中华麻醉学杂志

Objective:To evaluate the relationship between CCL21 and triggering receptor expressed on myeloid cells 2 (TREM2)/DNAX-activating protein of 12 kDa (DAP12) signaling pathways in the spinal dorsal horn in remifentanil-induced hyperalgesia in mice with incisional pain.Methods:Thirty-two SPF healthy male C57BL/6J mice, weighing 18-22 g, aged 8-10 weeks, were divided into 4 groups ( n=8 each) using a random number table method: control group (group C), CCL21 neutralizing antibody group (group anti-CCL21), remifentanil + incisional pain group (group R+ I), and CCL21 neutralizing antibody + remifentanil + incisional pain group (group anti-CCL21+ R+ I).A CCL21 neutralizing antibody 0.3 μg (diluted to 10 μl in normal saline) was intrathecally injected in anti-CCL21 and anti-CCL21+ R+ I groups twice a day.Normal saline 10 μl was intrathecally injected at the same time point twice a day in C and R+ I groups.Fifteen min after intrathecal injection, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at an interval of 15 min in C and anti-CCL21 groups, and remifentanil 10 μg/kg (diluted to 0.1 ml in normal saline) was injected via the caudal vein for 4 consecutive times at an interval of 15 min in R+ I and anti-CCL21+ R+ I groups.The tail-flick latency (TFL) and mechanical paw withdrawal threshold (MWT) were measured at 24 h before remifentanil or normal saline injection (T 0) and 3, 6, 24 and 48 h after stopping injection of remifentanil or normal saline (T 1-4).The mice were sacrificed after the last measurement of pain threshold, and L 4-6 segments of the spinal cord were removed for determination of the expression of TREM2 and DAP12 protein and mRNA (by Western blot or quantitative real-time polymerase chain reaction). Results:Compared with group C, TFL was significantly shortened and MWT was decreased at T 1-4, and the expression of TREM2 and DAP12 protein and mRNA was up-regulated in group R+ I and R+ I+ anti-CCL21 ( P<0.05), and no significant change was found in the parameters mentioned above in group anti-CCL21 ( P>0.05).Compared with group R+ I, TFL was significantly prolonged and MWT was increased at T 1-4, and the expression of TREM2 and DAP12 protein and mRNA was down-regulated in group anti-CCL21+ R+ I ( P<0.05). Conclusions:CCL21 is involved in remifentanil-induced hyperalgesia by activating TREM2/DAP12 signaling pathways in the spinal dorsal horn of mice with incisional pain.

Expression of serum soluble triggering receptor expressed on myeloid cells 2 in patients with vascular dementia and its relationship with β-amyloid 1-42 and lipoprotein-associated phospholipase 2 / 中华老年医学杂志

Objective:To investigate the expression levels of serum soluble triggering receptor expressed on myeloid cells 2(sTREM2)in patients with vascular dementia(VD)and its relationship with β-amyloid 1-42(Aβ1-42)and lipoprotein-associated phospholipase 2(Lp-PLA2), as well as its value in diagnosis and differential diagnosis.Methods:A total of 152 patients with ischemic stroke receiving routine treatment in our hospital from January 2018 to January 2020 were included and divided into a VD group(n=76)and a non-VD group(n=76)according to evaluation from subsequent visits at 3 months.General data, Mini-Mental State Examination Scale(MMSE), Hachinski Ischemic Scale(HIS)scores, and the levels of biochemical indicators were compared and analyzed for the two groups.Results:There were statistically significant differences in lesion size between the VD group and the non-VD group( P<0.05). The MMSE score in the VD group was significantly lower than that in the non-VD group.The HIS score, serum levels of sTREM2[(3.34±1.18)μg/L and(2.78±1.25)μg/L, t=2.121, P=0.036], Aβ1-42[(93.69±14.45)ng/L and(81.24±14.21)ng/L, t=4.003, P<0.001]and Lp-PLA2 levels[(58.67±14.15)μg/L and(43.18±13.86)μg/L, t=5.096, P<0.001]were significantly higher in patients with VD than in patients without VD( P<0.05). Serum sTREM2 was positively correlated with Aβ1-42( r=0.723, P<0.001)and Lp-PLA2( r=0.714, P<0.001), and Aβ1-42 was positively correlated with Lp-PLA2( r=0.698, P<0.001)in VD patients.The cut-off value, sensitivity, specificity, and area under the curve of serum sTREM2 for differentiating VD from non-VD were 3.96 μg/L, 81.30%, 78.40%, and 0.838, respectively. Conclusions:Serum sTREM2 is abnormally elevated in VD patients, and is significantly correlated with Aβ1-42 and LP-PLA2, thus STREM2 may be an indicator in the differential diagnosis of VD and non-VD.

Desencadeamento na neurose e sua relação com o sinthoma e a função do analista no contexto da clínica na atualidade / The concept of triggering in neurosis and its relationship with sinthome and the role of the psychoanalyst in the context of current clinical psychology / Le concept de déclenchement dans la névrose et son rapport avec le sinthome et le rôle du psychanalyste dans le contexte de la psychologie clinique actuelle) / El concepto de desencadenamiento en la neurosis y su relación con el sinthome y el papel del psicoanalista en el contexto de la psicología clínica actual)

Este trabalho aborda a noção de desencadeamento nas neuroses, considerando a clínica atual e, para isto, perpassa pela noção de encadeamento e desencadeamento considerando ensinamentos freudiano e lacaniano. Formalizações sobre sinthoma, como elemento que repara os erros do enlaçamento e índice de encadeamento, e sintoma, são convocadas para a conversa em que a neurose, mesmo no contexto da clínica, apresenta-se frequentemente em sua forma não desencadeada. A função do analista como perturbador da defesa e do sinthoma é, assim, questão que se coloca em cena.

This article addresses the concept of triggering in neurosis by taking into account current clinical psychology and based on the concepts of chaining and triggering, according to Freudian and Lacanian teachings. Formalizations about the sinthome as an element that repairs bonding mistakes and the chaining index, and the sinthome itself, are convened for a conversation in which neurosis, even in the context of clinical psychology, often presents itself in its non-triggered form. The role of the psychoanalyst as a disrupter of both defense and the sinthome is therefore a question that arises in this scenario.

Cette étude examine la notion de déclenchement dans la névrose en prenant pour base la psychologie clinique actuelle ainsi que les concepts de chaînage et de déclenchement selon les enseignements freudiens et lacaniens. Les formalisations sur le sinthome, en tant qu'élément qui répare les erreurs de liaison et de l'index de chaînage, ainsi que le sinthome, sont convoqués à la conversation dans laquelle la névrose, même dans le contexte de la psychologie clinique, se présente souvent sous sa forme non-déclenchée. Le rôle du psychanalyste en tant que perturbateur de la défense et du sinthome représente donc une question qui se met en évidence.

Este trabajo aborda el concepto de desencadenamiento en las neurosis, considerando la práctica clínica actual y, para ello, transita por los conceptos de encadenamiento y desencadenamiento, basándose en las enseñanzas freudianas y lacanianas. Las formalizaciones sobre el sinthome, como un elemento que repara los errores de cruce y el índice de encadenamiento, y el síntoma en sí, son convocados a la charla en la que la neurosis, incluso en el contexto de la práctica clínica, se presenta a menudo en su forma no desencadenada. El papel del psicoanalista como perturbador, tanto de la defensa como del sinthome es, por lo tanto, una cuestión que surge en la escena.

Role and regulatory mechanism of triggering receptor expressed on myeloid cells 2 in mice lung ischemia/reperfusion injury / 中华危重病急救医学

Objective:To investigate the role and regulatory mechanism of triggering receptor expressed on myeloid cell 2 (TREM2) in mice lung ischemia/reperfusion injury (LIRI).Methods:Thirty-six healthy male C57BL/6 mice were divided into six groups according to the random number method ( n = 6): normal control group, and LIRI 2, 6, 12, 24, 48 hours group. Mice LIRI models were established by clamping the left hilum. The wet/dry weight ratio (W/D) of left lung tissue was measured. Lung injury was observed and evaluated by hematoxylin-eosin (HE) staining and electron microscopy. The levels of interleukins (IL-1β, IL-18) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of TREM2 and caspase-1 were determined by polymerase chain reaction (PCR). The protein expressions of TREM2, caspase-1, Gasdermin-D (GSDMD) were determined by Western blotting. Results:At 2 hours after LIRI, lung injury began to appear, the lung ultrastructure changed, and the lung injury score increased; at 6 hours, the degree of lung injury was the most serious; after 12 hours, the lung injury gradually reduced and the lung injury score gradually decreased. Compared with the normal control group, lung W/D ratio and lung injury score of LIRI 2, 6, 12, 24, 48 hours groups were significantly higher, the differences were statistically significant (lung W/D ratio: 7.06±0.52, 8.34±0.17, 6.42±0.35, 5.34±0.25, 5.59±0.45 vs. 4.69±0.23; lung injury score: 5.50±0.54, 9.75±0.89, 5.88±0.84, 3.63±0.74, 4.13±0.64 vs. 1.13±0.35, all P < 0.05). Compared with the normal control group, the levels of IL-1β and IL-18 in lung tissue were significantly increased at 2 hours after LIRI, reached a peak at 6 hours [IL-1β (ng/L): 502.76±12.25 vs. 56.50±8.07, IL-18 (ng/L): 414.02±10.75 vs. 81.63±5.29, both P < 0.05], then decreased gradually, and were still significantly higher than the normal control group at 48 hours. The PCR and Western blotting showed that the expression of TREM2 was significantly lower than that in the normal control group at 2 hours after LIRI, and reached a valley at 6 hours [TREM2 mRNA (2 -ΔΔCt): 0.47±0.05 vs. 1.02±0.05, TREM2/GAPDH: 0.23±0.13 vs. 0.48±0.17, both P < 0.05], then gradually increased, and reached the peak at 24 hours [TREM2 mRNA (2 -ΔΔCt): 3.98±0.15 vs. 1.02±0.05, TREM2/GAPDH: 0.71±0.17 vs. 0.48±0.17, both P < 0.05]. The trend of expression of caspase-1 and GSDMD were opposite to that of TREM2, which increased at first and then decreased, and reached a peak at 6 hours after reperfusion [caspase-1 mRNA (2 -ΔΔCt): 2.20±0.13 vs. 1.01±0.02, caspase-1/GAPDH: 0.64±0.02 vs. 0.20±0.06, GSDMD/GAPDH: 1.23±0.01 vs. 0.87±0.02, all P < 0.05]. Conclusions:TREM2 might be involved in LIRI in mice. The mechanism may be related to the effect of TREM2 on caspase-1-mediated pyroptosis.

Pendelluft volume during double-triggered asynchronous breaths under pressure support ventilation: a prospective physiological study / 中华危重病急救医学

Objective:To investigate the relationship between double-triggering and abnormal movement of air in the lungs (pendelluft phenomenon) under pressure support ventilation (PSV).Methods:A prospective observational study was conducted, postoperative patients admitted to department of critical care medicine of Beijing Tiantan Hospital, Capital Medical University from April 1, 2019 to August 31, 2020 and received invasive mechanical ventilation with PSV mode were enrolled. Electrical impedance tomography (EIT) monitoring was performed. Airway pressure-time, flow-time, global and regional impedance-time curves were synchronously collected and analyzed offline. The volume of abnormal movement of air in the lungs at the beginning of inspiration was measured and defined as pendelluft volume. Double-triggered breaths were identified by trained researchers. Pendelluft volume during double-triggering was measured including the first triggered breath, the double-triggered breath, and the breath immediately following the double-triggered breath. Pendelluft volume was also measured for normal breath during the study. According to the frequency of double-triggering, patients were divided into severe (≥1 time/min) and non-severe double-triggering group. Pendelluft volume, parameters of respiratory mechanics, and clinical outcomes between the two groups were compared.Results:In 40 enrolled patients, a total of 9 711 breaths [(243±63) breaths/patient] were collected and analyzed, among which 222 breaths (2.3%) were identified as double-triggering. The Kappa of interobserver reliability to detect double-triggering was 0.964 [95% confidence interval (95% CI) was 0.946-0.982]. In 222 double-triggered breaths, pendelluft volume could not be measured in 7 breaths (3.2%), but the pendelluft phenomenon did exist as shown by opposite regional impedance change at the beginning of double-triggered inspiration. Finally, pendelluft volume was measured in 215 double-triggered breaths. Meanwhile, 400 normal breaths (10 normal breaths randomly selected for each patient) were identified as control. Compared with normal breath, pendelluft volume significantly increased in the first breath, the double-triggered breath, and the following normal breath [mL: 3.0 (1.4, 6.4), 8.3 (3.6, 13.2), 4.3 (1.9, 9.1) vs. 1.4 (0.7, 2.8), all P < 0.05]. Patients in severe double-triggering, pendelluft volume of normal breath and double-triggered breath were significantly higher than those in non-severe double-triggering group [mL: 1.8 (0.9, 3.2) vs. 1.1 (0.5, 2.1), P < 0.001; 8.5 (3.9, 13.4) vs. 2.0 (0.6, 9.1), P = 0.008]. Patients in severe double-triggering group had significantly higher respiratory rate than that in the non-severe double-triggering group (breaths/min: 20.9±3.5 vs. 15.2±3.7, P < 0.001). There were no significant differences in other respiratory mechanics parameters and main clinical outcomes between the two groups. Conclusions:During PSV, the abnormal movement of air in the lungs (pendelluft phenomenon) was more likely to occur in double-triggering especially in double-triggered breath. The more frequent the double-triggering occurred, the more serious the pendelluft phenomenon was. A higher pendelluft volume of normal breath and a higher respiratory rate were related to severity of double-triggering.

Role of TIPE2 in endogenous protective mechanism of acute lung injury in septic mice: relationship with TREM-1/NLRP3 inflammasome signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of tumor necrosis factor-alpha-induced protein-8 like-2 (TIPE2) in endogenous protective mechanism of acute lung injury (ALI) in septic mice and the relationship with triggering receptor expressed on myeloid cells-1 (TREM-1)/NOD-like receptor protein 3 (NLRP3) inflammasome signaling pathway.Methods:Twenty male wild-type mice and 20 TIPE2 knockout mice were divided into 4 groups using a random number table method: wild-type+ sham operation group (group WT-sham), wild-type+ ALI group (group WT-ALI), TIPE2-knockout+ sham operation group (group KO-sham) and TIPE2-knockout+ CLP group (group KO-ALI), with 10 mice in each group.The ALI model was established by cecal ligation and perforation (CLP) in septic mice.Mice were sacrificed after blood samples were obtained from the abdominal aorta at 24 h after CLP, and lung tissue specimens were obtained for microscopic examination of pathological changes (with a light microscope) which were scored and for determination of wet/dry weight ratio (W/D ratio), myeloperoxidase (MPO) activity, expression of TIPE2, TREM-1, NLRP3, caspase-1 and GSDMD (by Western blot), and concentrations of interleukin-1beta (IL-1β) and IL-18 in serum (by enzyme-linked immunosorbent assay).Results:Compared with group WT-sham, the lung injury score, W/D ratio, MPO activity and concentrations of IL-1β and IL-18 in serum were significantly increased, the expression of TREM-1, NLRP3, caspase-1 and GSDMD was up-regulated, and the expression of TIPE2 was down-regulated in group WT-ALI and group KO-ALI ( P<0.05). Compared with group WT-ALI, the lung injury score, W/D ratio, MPO activity and concentrations of IL-1β and IL-18 in serum were significantly increased, the expression of TREM-1, NLRP3, caspase-1 and GSDMD was up-regulated, and the expression of TIPE2 was down-regulated in group KO-ALI ( P<0.05). Conclusion:TIPE2 is involved in endogenous protective mechanism of ALI in septic mice, which is related to inhibition of activation of TREM-1/NLRP3 inflammasome signaling pathway.

Effect and mechanism of monocytes/macrophages TREM-1 on acute lung injury in rats with severe heatstroke / 解放军医学杂志

Objective: To investigate the effect of the monocytes/macrophages on acute lung injury in rats with severe heatstroke, by modulating the expression of triggering receptor expressed on myeloid cells-1 (TREM-1). Methods: Forty rats were randomized evenly into the control group (Con group), heatstroke group (HS group), the low dose inhibitor group (LD group) and the high dose inhibitor group (HD group). Before heatstroke induction, the rats of LD and HD groups were administrated with a 50 mg/kg and 100 mg/kg bolus of LP-17, respectively. All rats were exposed to an environment with temperature of (40 ± 2) °C and humidity of 65% ± 5% for 60 minutes to induce heatstroke. Enzyme-linked immunosorbent assay (ELISA) was utilized to quantify the concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β in the peripheral blood and pulmonary tissue. The expression of TREM-1 on peripheral monocytes was identified by flow cytometry. Moreover, the histological phenotypes were evaluated after HE stain and the expression of inducible nitric oxide synthase (iNOS) was analyzed by immunohistochemistry in pulmonary tissues. Furthermore, Western blotting was used to detect the protein level of TREM-1 and monocyte chemoattractant protein-1 (MCP-1). Results: Compared to HS rat, in rats pretreated with LP-17, the levels of TNF-α, IL-6 and IL-1β in peripheral blood (P<0.01) and pulmonary tissue (P<0.01) were descended; the upregulation of TREM-1 on peripheral monocytes was alleviated in (P<0.01); the histological injury (P<0.01) were reduced; the protein levels of iNOS, TREM-1 and MCP-1 (P<0.01) were down-regulated. Conclusion: The down-regulation of the TREM-1 activity on the monocytes/macrophages in the peripheral blood and lung tissue by the bolus of LP-17 benefit to ameliorate the lung injury induced by heatstroke via inhibiting inflammation, oxidative stress and chemokine.

Evaluation of the effect of intraperitoneal etanercept administration on oxidative stress and inflammation indicators in the kidney and blood of experimental sepsis-induced rats

Abstract INTRODUCTION: Sepsis is an important cause of mortality and morbidity, and inflammatory response and oxidative stress play major roles underlying its pathophysiology. Here, we evaluated the effect of intraperitoneal etanercept administration on oxidative stress and inflammation indicators in the kidney and blood of experimental sepsis-induced rats. METHODS: Twenty-eight adult Sprague Dawley rats were classified into Control (Group 1), Sepsis (Group 2), Sepsis+Cefazolin (Group 3), and Sepsis+Cefazolin+Etanercept (Group 4) groups. Kidney tissue and serum samples were obtained for biochemical and histopathological investigations and examined for the C reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), triggering receptor expressed on myeloid cells (TREM), and malondialdehyde (MDA) levels. RESULTS: The levels of TNF-α, TREM, and MDA in serum and kidney samples were significantly higher in rats from sepsis group than in rats from control group (p < 0.05). Group 3 showed a significant reduction in serum levels of TNF-α, CRP, and TREM as compared with Group 2 (p < 0.05). Serum TNF-α, CRP, TREM, and MDA levels and kidney TNF-α and TREM levels were significantly lower in Group 4 than in Group 2 (p < 0.05). Serum TNF-α and TREM levels in Group 4 were significantly lower than those in Group 3, and histopathological scores were significantly lower in Group 3 and Group 4 than in Group 2 (p < 0.05). Histopathological scores of Group 4 were significantly lower than those of Group 3 (p < 0.05). CONCLUSIONS: Etanercept, a TNF-α inhibitor, may ameliorate sepsis-induced oxidative stress, inflammation, and histopathological damage.

A elaboração conceitual da teoria freudiana do desencadeamento e estabilização / The conceptual elaboration of freud's theory of triggering and stabilization / La elaboración conceptual de la teoría freudiana del desencadenamiento y estabilización / L'élaboration conceptuelle de la théorie de freud sur le déclenchement et la stabilisation

O ponto de partida do artigo é mostrar a relevância e a atualidade da teoria freudiana da psicose. Nesse sentido, o objetivo é retomar a literatura visando a delimitar uma hipótese inaugural sobre o desencadeamento e a estabilização, em que a perturbação do funcionamento pulsional já se constituía como fator importante na etiologia psíquica de seu desencadeamento e o delírio, como um trabalho psíquico de reparação, de estabilização. Para isso, o artigo expõe um levantamento das referências freudianas a fim de mostrar o desenvolvimento epistemológico de uma pesquisa etiológica cuja especificidade reside em: 1. Considerar a etiologia da psicose à luz da invasão pulsional; 2. Isolar, por meio do conceito de rejeição, um processo psíquico específico de defesa contra a pulsão cujo resultado é a perda da realidade; 3. Definir a função do delírio como reparador, restituidor da realidade.

The starting point of the paper is to show the relevance and timeliness of Freud's theory of psychosis. In this sense, the objective is to retake the literature aiming to delimit an inaugural hypothesis about the triggering and the stabilization, in which the disturbance of the drive functioning was already constituted as an important factor in the psychic etiology of its triggering and the delirium, as a psychic work repair, stabilization. To this end, the article exposes a survey of Freudian references to show the epistemological development of the etiological research whose specificity lies in 1: Considering the etiology of psychosis in light of the drive invasion; 2: Isolate, through the concept of rejection, a specific psychic process of defense against the drive which results in the loss of reality; and 3: Define the function of delirium as restorative, a restorer of reality.

El punto de salida de este trabajo es enseñar la relevancia y la actualidad de la teoría freudiana de la psicosis. En este sentido, el objetivo es retomar la literatura para delimitar una hipótesis inaugural sobre el desencadenamiento y la estabilización, en que la perturbación del funcionamiento pulsional ya se constituía como factor importante en la etiología psíquica de su desencadenamiento y el delirio, como un trabajo psíquico de reparación, de estabilización. Para eso, el trabajo expone una búsqueda de las referencias freudianas con el fin de enseñar el desenvolvimiento epistemológico de una investigación etiológica cuya especificidad reside en: 1. Considerar la etiología de la psicosis a la luz de la invasión pulsional; 2. Aislar, por medio del concepto de rechazo, un proceso químico específico de defensa contra la pulsión cuyo resultado es la perdida de la realidad; 3. Definir la función del delirio como reparador, restituidor de la realidad.

Le point de départ de cet article est de montrer la pertinence et l'actualité de la théorie de Freud sur la psychose. En ce sens, l'objectif est de reprendre la littérature pour délimiter une hypothèse inaugurale sur le déclenchement et la stabilisation. Dans l'hypothèse, le dérangement du fonctionnement de la pulsion était déjà constitué comme un facteur important dans l'étiologie psychique de son déclenchement. Du délire, à son tour, comme un travail psychique de réparation et de stabilisation. Pour cela, l'article expose des références freudiennes afin de montrer le développement épistémologique d'une recherche étiologique dont la spécificité réside dans: 1. Considérer l'étiologie de la psychose à la lumière de désintrication pulsionnelle; 2 Isoler, à travers le concept de rejet, un processus psychique spécifique de défense contre la pulsion dont le résultat et la perte de la réalité; 3 Définir la fonction du délire en tant que réparateur, restaurateur de la réalité.

Triggering Factorsfor Breakthrough Seizures among Epileptic Patients. Cross Sectional Study in Epileptic Patients Attending Epilepsy Clinic Mansoura University Hospital, Egypt

Background: The prevalence of breakthrough seizures in persons with epilepsyis very high in developing countries. Consequently, patients and physicians should be aware of the possible factors that may cause breakthrough seizures.Objective: The aim of our study is to determine the possible factors that may be a precipitating cause for breakthrough seizures in patients with epilepsy.Methods: This cross-sectional study included 100 persons with epilepsywith idiopathic epilepsy receiving antiepileptic drugs (AEDs). They were divided into two groups. Group 1 included 50 persons with epilepsywith a history of recent breakthrough seizures. Group 2 included 50 persons with epilepsywho had not experienced any recent breakthrough seizures. Patients were subjected to a thorough questionnaire addressing precipitating factors. All participants were subjected to an electroencephalogram (EEG) and medication adherence assessment.Results: There was no significant differences between group 1 and group 2 regarding age, sex, ageOriginal Research Article of onset of epilepsy, occupation and marital status (P value range 0.5 –0.2). The patients in group 1 were found to have longer durations of epilepsy, lower adherence to AEDs (P= 0.001), moremissed doses of AEDs (P= 0.0001), more side effects of AEDs (P = 0.0005), more sleep deprivation, lower level of AEDs (P= 0.0006), more frequently on AED polytherapy (P = 0.0002), and more flickering lights(P= 0.04) than the participants in group 2. In terms of the EEG, group 1 showed a higher percentage of abnormal EEGs and more frequent focal epileptiform discharges (P = 0.003). Also, pathological findings in MRI brain were associated with higher breakthrough seizures (P = 0.005). No significant difference was found in both group1 and group 2 regarding emotional stress (P = 0.55), substitution of brand AEDs by generic one (P = 0.83), concurrent illness (P = 1), or the use of non AEDs (P = 0.79). Conclusion: The precipitating factors of breakthrough seizures are multifactorial and it is very important to educate patients about these precipitating factors to achieve better control of epilepsy

Diagrama de Ishikawa: causas e soluções da infecção ao HIV adquirida por profissionais de enfermagem através de materiais perfurocortantes / Ishikawa Diagram: causes and solutions of HIV infection in nursing professionals due to sharps use / Diagrama de Ishikawa: causas y soluciones de la infección al vih adquirida por profesionales de enfermado a través de materiales perfurocortantes

Objective: The study's goal has been to analyze, through the Ishikawa Diagram, the causes and solutions of HIV infection in nursing professionals due to sharps handling. Methods: It is a literature review based on the Construction of the Ishikawa Diagram, which was carried out in May 2017 in the PubMed and Virtual Health Library databases. Results: The following are the main causes of HIV infection through sharps: work overload, perception of frail risk, careless use of needles, lack of training. Regarding the solutions: adequate post-exposure management, implementing and monitoring compliance with biosafety standards, improving the notification of accidents with sharps. Conclusion: There is a need to alerting managers towards intervening in the factors that might trigger accidents with sharp materials by the nursing team

Objetivo: Analisar por meio do Diagrama de Ishikawa as causas e soluções da infecção ao HIV adquirida por profissionais de enfermagem no manuseio de materiais perfurocortantes. Métodos: Revisão da literatura baseada na Construção do Diagrama de Ishikawa, realizado em maio de 2017 nas bases de dados Pubmed e Biblioteca Virtual de Saúde. Resultados: Dentre as causas da infecção ao HIV através de perfurocortantes: sobrecarga de trabalho, percepção de risco fragilizada, utilização descuidada de agulhas, ausência de treinamento. Quanto às soluções: gestão pós exposição adequada, implementar e fiscalizar o cumprimento das normas de biossegurança, aprimorar a notificação de acidentes com materiais perfurocortantes. Conclusão: Destaca-se a necessidade de sensibilizar gestores para intervir nos fatores que podem desencadear acidentes com materiais perfurocortantes pela equipe de enfermagem

Objetivo: Analizar por medio del Diagrama de Ishikawa las causas y soluciones de la infección al VIH adquirida por profesionales de enfermería en el manejo de materiales punzocortantes. Métodos: Revisión de la literatura basada en la construcción del diagrama de Ishikawa, realizado en mayo de 2017 en las bases de datos Pubmed y Biblioteca Virtual de Salud.Resultados: Entre las causas de la infección por el VIH a través de punzocortantes: sobrecarga de trabajo, percepción de riesgo fragilizada, utilización descuidada de agujas, ausencia de entrenamiento. En cuanto a las soluciones: gestión post exposición adecuada, implementar y fiscalizar el cumplimiento de las normas de bioseguridad, mejorar la notificación de accidentes con materiales punzocortantes. Conclusión: Se destaca la necesidad de sensibilizar a los gestores para intervenir en los factores que pueden desencadenar accidentes con materiales punzocortantes por el equipo de enfermería

An observational study to assess the knowledge of migraine among patients with migraine at a tertiary care teaching hospital

Background: Migraine is a common debilitating headache the exact cause of which is not known. Efficient and proper treatment of migraine involves accurate diagnosis, educating the patients and to make them aware of the disorder and finding the best medication for the management of pain. This will also increases the patients� adherence to medication and will help in improving their quality of life. The objective of the study was to assess the awareness of migraine patients towards various aspects of migraine in a tertiary care teaching hospital.Methods: This was a cross-sectional observational questionnaire-based study conducted from January to March 2017 in ASCOMS&H, Sidhra, Jammu. The self-administered pre-validated questionnaires pertaining to various aspects of migraine were distributed among the patients diagnosed of having migraine and attending the Neurology Out patient department (OPD). Patients of either sex with a diagnosis of migraine and age ?18 years were included in the study. The data was analyzed with the help of descriptive statistics.Results: In the present study, majority of the patients suffering from migraine were in the age group of 18-40 years (58.6%) and females were more commonly affected about 60.9% than males. There was no family history of migraine in 78.1% of the patients. About 87.4% of the patients had 2-6 attacks of migraine per month. The most common triggering factor cited by the patients was lack of sleep in 29.9% followed by stress in 27.6% and less water intake in 26.4% of the patients.Conclusions: From the present study, it can be concluded that the patients were aware about the associated signs and symptoms but they had inadequate knowledge about all the aspects of the disease. Therefore, it is important to make patients more aware of the disease to increase their compliance to treatment and to improve their quality of life.

Effects of Dexmedetomidine Combined with Mild Hypothermia on the Levels of HMGB 1，TLR4 and Trem- 1 in Lung Tissues of Sepsis Model Rats / 中国药房

OBJECTIVE： To investigate the effects of dexmedetomidine combined with mild hypothermia on the levels of High mobility group box 1 protein （HMGB1）， Toll like receptor 4 （TLR4） and Triggering receptor expressed on myeloid cells 1 （Trem-1） in lung tissues of sepsis model rats. METHODS： Totally 100 SD rats were randomly divided into sham operation group （normal saline）， model group （normal saline）， dexmedetomidine group （2 μg/kg）， mild hypothermia group （normal saline+anal temperature 32-35 ℃ caused by whole body spraying of cold water） and combination group （dexmedetomidine 2 μg/kg+anal temperature 32-35 ℃ caused by whole body spraying of cold water）， with 20 rats in each group. Except that sham operation group received sham operation， sepsis model was induced in other groups. After ligation and incision， the corresponding drugs were pumped into the jugular vein and the corresponding body temperature was maintained. Plasma samples were collected 6 h after operation. Interleukin 1 （IL-1）， IL-6， tumor necrosis factor α （TNF-α） were determined by ELISA. The lung wet mass/dry mass ratio （W/D） was calculated by weighing the mass. Lung tissue sections were observed by HE staining， and lung injury scores were scored. The activity of MPO in lung tissue was determined by immunoturbidimetry. The expression of HMGB1，TLR4 and Trem-1 protein was determined by Western blot. RESULTS： Compared with sham operation group， the contents of IL-1， IL-6 and TNF-α， W/D， lung injury score， MPO activity， the protein expression of HMGB1， TLR4 and Trem-1 were increased significantly， with statistical significance （P＜0.05）； lung tissue section showed that alveolar wall was obviously thickened； a large number of inflammatory cells infiltrated； blood vessels were obviously dilated and congested. Compared with model group， above indexes of rats in dexmedetomidine group， mild hypothermia group and combination group were decreased significantly， with statistical significance （P＜0.05）； the degree of pathological changes in lung tissue was significantly reduced. Compared with dexmedetomidine group and mild hypothermia group， above indexes of combination group were decreased more significantly， with statistical significance （P＜0.05）. Alveolar walls were thickened， inflammatory cell infiltration was relieved significantly and no vascular diatation and congestion was found. CONCLUSIONS： Dexmedetomidine combined with mild hypothermia can significantly improve lung injury in sepsis model rats， and down-regulate the protein expression of HMGB1， TLR4 and Trem-1. Therapeutic efficacy of combination therapy is better than single therapy.

Effects and mechanism of TREM-1 On inflammatory response and lipid metabolism in mice with non-alcoholic fatty liver disease / 中华肝胆外科杂志

0bjective Analysis of the effect of triggering receptor-1 expressed on myeloid cells(TREM-1)in nonalcoholic fatty liver disease(NAFLD)and the mechanism.Methods The oleic acid-treated HepG2 ceils were divided into model group,overexpression group,interference group A,interference group B and negative control group.The mouse model of NAFLD was generated and randomly divided into(nuclear factor-κB)NF-κB inhibition group,protein kinase B(AKT)inhibition group,knockout group A,knockout group B and control group.The expression of inflammatory factors and TREM.1 in liver tissue was detected by PCR,and fat accumulation was detected by oil red O staining.Western blotting was used to detect the expression of TREM-1 and signaling pathway proteins,and HE staining was used to detect liver tissue changes.Results TREM-1 was up.regulated in liver tissue of NAFLD mice J(0.936±0.127)vs.(0.432±0.105)] and in oleic acid-treated HepG2 cells.In oleic acid.treated HepG2 cells.overexpression of TREM-1 increased inflammatory factor expression and increased 1ipid droplets:inhibition of TREM-1 expression decreased inflammatory factor expression.and lipid droplets decreased.Knockout of TREM-1 and inhibition of NF-κB in NAFLD mice reduced hepatocyte inflammatory factor expression and reduced liver damage;knockout of TREM-1 and inhibition of AKT reduced liver tissue lipids and drops accumulate,Conclusions The overexpression of TREM-1 in NAFLD mice liver tissue can regulate inflammatory factor expression and lipid droplets through NF-κB and AKT signal pathway.TREM-1 might be a potential thera-peutic target of NAFLD.

Effects and mechanism of TREM-1 on inflammatory response and lipid metabolism in mice with nonalcoholic fatty liver disease / 中华肝胆外科杂志

Objective@#Analysis of the effect of triggering receptor-1 expressed on myeloid cells (TREM-1) in nonalcoholic fatty liver disease (NAFLD) and the mechanism.@*Methods@#The oleic acid-treated HepG2 cells were divided into model group, overexpression group, interference group A, interference group B and negative control group. The mouse model of NAFLD was generated and randomly divided into (nuclear factor-κB) NF-κB inhibition group, protein kinase B (AKT) inhibition group, knockout group A, knockout group B and control group. The expression of inflammatory factors and TREM-1 in liver tissue was detected by PCR, and fat accumulation was detected by oil red O staining. Western blotting was used to detect the expression of TREM-1 and signaling pathway proteins, and HE staining was used to detect liver tissue changes.@*Results@#TREM-1 was up-regulated in liver tissue of NAFLD mice [(0.936±0.127) vs. (0.432±0.105)] and in oleic acid-treated HepG2 cells. In oleic acid-treated HepG2 cells, overexpression of TREM-1 increased inflammatory factor expression and increased lipid droplets; inhibition of TREM-1 expression decreased inflammatory factor expression, and lipid droplets decreased. Knockout of TREM-1 and inhibition of NF-κB in NAFLD mice reduced hepatocyte inflammatory factor expression and reduced liver damage; knockout of TREM-1 and inhibition of AKT reduced liver tissue lipids and drops accumulate.@*Conclusions@#The overexpression of TREM-1 in NAFLD mice liver tissue can regulate inflammatory factor expression and lipid droplets through NF-κB and AKT signal pathway. TREM-1 might be a potential therapeutic target of NAFLD.

Effect of Dahuang Mudan Tang in Triggering Receptor Expressed on Myeloid Cells-1 in Intestinal Tract in Rats with Septic Acute Intestinal Dysfunction / 中国实验方剂学杂志

Objective:To investigate whether the therapeutic effect of Dahuang Mudan Tang on septic acute intestinal dysfunction in sepsis ratsis related to the regulation of expression of triggering receptor expressed on myeloid cells-1(TREM-1). Method:Totally 100 male SD rats were injected intraperitoneally with lipopolysaccharide (LPS) at a dose of 4.5 mg·kg-1 to build sepsis model. The sepsis model rats were randomly divided into five groups:model group, glutamine group (3.75 g·kg-1),low,medium, high-dose Dahuang Mudan Tang group(7.5,15,30 g·kg-1),and another 10 normal rats were selected as normal group. Seven days later,2 mL suspension (100 mg lactulose and 50 mg mannitol) was orally administrated by gavage, and 24 h urinewas collected. The ratio of lactulose to mannitol in urine (L/M) was detected by HPLC with pulsed electrochemical detection (HPLC-PED).Serum citrulline concentrationsin blood and ileum were determined by HPLC.Enzyme linked immunesorbent assay (ELISA) was used to detect the concentrations of triggering receptor expressed on myeloid cells-1(TREM-1),tumor necrosis factor-α(TNF-α),intestinal fatty acid binding protein(iFABP) and D-lactic acid.Real-time PCR was used to detect the mRNA expressions of TREM-1,Toll-like receptors2(TLR2),Toll-like receptors 4(TLR4),myeloid cell differentiation protein(MyD88),nuclear transcription factor-κB(NF-κB).Electron microscopy was used to observe the pathological changes of intestinal mucosa injury. Result:Compared with normal group, the serum concentrations of TREM-1,TNF-α,iFABP, D-lactate; the ratio of lactulose to mannitol in urine (L/M)and the expressions of TREM-1,TLR2,TLR4,MyD88,NF-κB mRNA in model group were increased obviously(PPPPPκB mRNA,and the serum concentrations of TREM-1 and TNF-α in all medication administration groups were decreased obviously(PD-lactate, L/M, the Chiu scorein glutamine group, medium-dose Dahuang Mudan Tang group and high-dose Dahuang Mudan Tang group were decreased obviously(PPPConclusion:Dahuang Mudan Tang can effectively treat SAID in rats, and its mechanism may be realized by regulating the expression of TREM-1 and relieving intestinal inflammation of intestinal tract.

Reason analysis of ineffective triggering caused by a continuous nebulization airflow in mechanical ventilation / 中华危重病急救医学

Objective? To? analyze? the? ineffective? triggering? caused? by? nebulization? in? the? way? of?respiratory?mechanics.? Methods? A?test-lung?and?an?oxygen-driven?jet?nebulizer?were?connected?to?the?circuit?in?a?PB840?ventilator.?The?test-lung?was?pulled?outwards?in?manual?way?till?an?inspiration?was?effectively?triggered?separately?in?different?flow-trigger?modes?[flow-trigger?sensitivity?(VTrig)?3?L/min?and?5?L/min]?and?pressure-trigger?modes?[pressure-trigger?sensitivity?(PTrig)?2?cmH2O?and?4?cmH2O,?1?cmH2O?=?0.098?kPa]?with?the?nebulizer?being?closed?and?opened?in?turn.?The?corresponding?relationship?and?characteristics?between?the?flow?and?pressure?in?the?circuit?under?different?triggering?conditions?were?observed?by?adjusting?the?curve?amplitude?in?the?screen.?The?minimum?alveolar?pressure?(Pa)?which?could?cause?an?effective?triggering?and?the?variation?span?of?Pa?during?the?triggering?period?were?analyzed?in?respiratory?mechanics.? Results? ①?In?flow-trigger?mode:?Pa?was?pulled?down? from? positive? end-expiratory? pressure? (PEEP)? or? intrinsic? positive? end-expiratory? pressure? (PEEPi)? to?"PEEP－VTrigR"?(R?meant?airway?resistance)?without?nebulization,?and?the?span?of?Pa?was?"VTrigR"?or?"PEEPi－PEEP+VTrigR".?Pa?was?pulled?down?from?PEEP?or?PEEPi?to?"PEEP－(VTrig+N)?R"?(N?meant?nebulization?airflow)?with?nebulization,?and?the?span?of?Pa?was?"(VTrig+N)?R"?or?"PEEPi－PEEP+(VTrig+N)?R".?②?In?pressure-trigger?mode:?Pa?was?pulled?down?from?PEEP?or?PEEPi?to?"PEEP－PTrig－1R"?without?nebulization,?and?the?span?of?Pa?was?"PTrig+1R"?or?"PEEPi－PEEP+PTrig+1R".?Pa?was?pulled?down?from?PEEP?or?PEEPi?to?"PEEP－PTrig－(N+1)?R"?with?nebulization,?and?the?span?of?Pa?was?"PTrig+(N+1)?R"?or?"PEEPi－PEEP+PTrig+(N+1)?R".? ?Conclusions? Nebulization?airflow?increases?the?difficulty?of?inspiratory?triggering?in?mechanical?ventilation.?PEEPi?makes?it?more?difficult.