BACKGROUND:Studies have shown that there is a close relationship between pyroptosis,inflammatory factors and osteoporosis.OBJECTIVE:To review the effects of pyroptosis and inflammatory factors on the pathogenesis of osteoporosis from the perspectives of osteogenic differentiation and osteoclastic differentiation,based on an overview of pyroptosis in relation to the interaction of relevant inflammatory factors.METHODS:The first author used the computer to search the literature published by each database until 2024,and searched CNKI,WanFang,VIP and PubMed databases with the search terms of"pyroptosis,inflammatory factors,osteoporosis,osteoblast,osteoclast,bone metabolism,signaling pathway,review"in Chinese and English.A total of 79 papers were finally included according to the inclusion criteria.RESULTS AND CONCLUSION:The progression of osteoporosis is closely related to inflammation,in which pyroptosis plays a key role.Immune cells induce pyroptosis through apoptosis pathway,promote the secretion of inflammatory factors such as interleukin-18,interleukin-1β and NLRP3,build an inflammatory immune microenvironment,and regulate bone metabolism through complex signaling pathways,resulting in enhanced bone absorption and reduced bone formation,thereby leading to osteoporosis.Previous studies have shown that inhibiting pyroptosis is anti-inflammatory and slows the progression of osteoporosis,and it has been shown to improve inflammatory bone loss in vitro and in animal models.At present,research on pyroptosis and osteoporosis is limited.On the one hand,the exact mechanism of osteoporosis and the pathogenesis of pyroptosis are unknown,and the specific pathways and regulatory mechanisms remain to be understood.On the other hand,therapeutic strategies targeting pyroptosis are still theoretical,not clinically proven,and drug side effects are unknown.In the future,the research focus is to further explore the pathogenesis,especially the mechanism of pyroptosis,identify potential therapeutic targets,further study the pyroptosis signaling pathway and Gasdermin protein,and develop new drugs to improve the therapeutic effect in patients with osteoporosis.

BACKGROUND:Studies have shown that there is a close relationship between pyroptosis,inflammatory factors and osteoporosis.OBJECTIVE:To review the effects of pyroptosis and inflammatory factors on the pathogenesis of osteoporosis from the perspectives of osteogenic differentiation and osteoclastic differentiation,based on an overview of pyroptosis in relation to the interaction of relevant inflammatory factors.METHODS:The first author used the computer to search the literature published by each database until 2024,and searched CNKI,WanFang,VIP and PubMed databases with the search terms of"pyroptosis,inflammatory factors,osteoporosis,osteoblast,osteoclast,bone metabolism,signaling pathway,review"in Chinese and English.A total of 79 papers were finally included according to the inclusion criteria.RESULTS AND CONCLUSION:The progression of osteoporosis is closely related to inflammation,in which pyroptosis plays a key role.Immune cells induce pyroptosis through apoptosis pathway,promote the secretion of inflammatory factors such as interleukin-18,interleukin-1β and NLRP3,build an inflammatory immune microenvironment,and regulate bone metabolism through complex signaling pathways,resulting in enhanced bone absorption and reduced bone formation,thereby leading to osteoporosis.Previous studies have shown that inhibiting pyroptosis is anti-inflammatory and slows the progression of osteoporosis,and it has been shown to improve inflammatory bone loss in vitro and in animal models.At present,research on pyroptosis and osteoporosis is limited.On the one hand,the exact mechanism of osteoporosis and the pathogenesis of pyroptosis are unknown,and the specific pathways and regulatory mechanisms remain to be understood.On the other hand,therapeutic strategies targeting pyroptosis are still theoretical,not clinically proven,and drug side effects are unknown.In the future,the research focus is to further explore the pathogenesis,especially the mechanism of pyroptosis,identify potential therapeutic targets,further study the pyroptosis signaling pathway and Gasdermin protein,and develop new drugs to improve the therapeutic effect in patients with osteoporosis.

BACKGROUND:It has also been confirmed that ferroptosis is closely related to a variety of musculoskeletal diseases,such as rheumatoid arthritis,osteosarcoma,and osteoporosis.The pathophysiological mechanisms of ferroptosis and osteoporosis need to be further studied and elucidated to broaden our understanding of iron metabolism and osteoporosis.It will provide research ideas for the future elucidation of new mechanisms of osteoporosis and the development of new technologies and drugs for the treatment of osteoporosis. OBJECTIVE:To provide an overview of the current status of research on ferroptosis in osteoporosis,to provide a new direction for future research on the specific molecular mechanisms of osteoporosis,and to provide more effective and better options for osteoporosis treatment strategies. METHODS:The first author used the computer to search the literature published from 2000 to 2024 in CNKI,WanFang,VIP,and PubMed databases with search terms"ferroptosis,iron metabolism,osteoporosis,osteoblast,osteoclast,bone metabolism,signal pathway,musculoskeletal,review"in Chinese and English.A total of 68 articles were finally included according to the selection criteria. RESULTS AND CONCLUSION:(1)Ferroptosis is a new type of cell death discovered in recent years,which is usually accompanied by a large amount of iron accumulation and lipid peroxidation during cell death,and its occurrence is iron-dependent.This is distinctly different from several types of cell death that are currently being hotly studied(e.g.,cellular pyroptosis,necrotic apoptosis,cuproptosis,and autophagy).(2)Intracellular iron homeostasis is manifested as a balance between iron uptake,export,utilization,and storage.The body's iron regulatory system includes systemic and intracellular regulation.The main factor of systemic regulation is hepcidin produced by hepatic secretion,and cellular regulation depends on the iron regulatory protein/iron response element system.Of course,intracellular iron homeostasis can be controlled by other factors,such as hypoxia,cytokines,and hormones.(3)Lipid peroxidation causes oxidative damage to biological membranes(plasma membrane and internal organelle membranes),lipoproteins,and other lipid-containing molecules.Polyunsaturated fatty acid-containing phospholipids are important targets of lipid peroxidation.Free polyunsaturated fatty acid is an important substrate for lipid oxidation and can bind to the phospholipid bilayer,leading to over-oxidation and thus triggering lipid apoptosis.(4)Several studies have shown that osteoblasts are overloaded with iron in different ways,resulting in the accumulation of unstable ferrous iron and the generation of reactive oxygen species and lipid peroxides,causing ferroptosis of osteoblasts and ultimately a decrease in bone formation,affecting bone homeostasis and the development of osteoporosis.(5)Osteoclasts are large multinucleated cells formed by the fusion of mononuclear macrophage cell lines or bone marrow mesenchymal stem cells induced by nuclear factor-κB ligand receptor activator,and they have the function of bone resorption.Iron ions can promote osteoclast differentiation and bone resorption through the production of intracellular lipid reactive oxygen species,while iron chelators can inhibit osteoclast formation in vitro and thus affect the occurrence and development of osteoporosis.

Objective To investigate the effect of the Nogo receptor antagonist NEP1-40,administered via an exogenous route,on axonal regeneration in rats with spinal cord injury(SCI),and to explore its mecha-nism of action in the process of neural repair.Methods SD rats were divided into a sham surgery group(Group A),an injury group(Group B),and an injury+NEP1-40 treatment group(Group C).In Group A,only laminectomy was performed without spinal cord injury.Groups B and C were subjected to a clip-type SCI model.Group C received treatment with NEP1-40 based on the established SCI model.Hindlimb motor func-tion in the three groups was assessed using the BBB score at 1,3,7,and 14 days post-surgery.Real-time quan-titative PCR(qPCR)and Western blot were used to detect changes in gene and protein expression levels of growth-associated protein-43(GAP-43)and microtubule-associated protein-2(MAP-2),characteristic markers of axonal and dendritic regeneration.Immunofluorescence was employed to analyze NF-200 and BrdU double-labeling,and changes in the number of double-labeled positive cells were observed and analyzed.Results In group A rats,the BBB scores at various time points after surgery showed no significant change compared with preoperative scores.In groups B and C,the BBB scores on postoperative day 1 were obviously lower than pre-operative scores.From days 3 to 14 after surgery,the BBB scores partially recovered compared with postopera-tive day 1,though they remained lower than those in group A.However,on postoperative days 3,7,and 14,the BBB scores in group C were higher than those in group B(P<0.05).qPCR and Western blot results showed that compared with preoperative levels,GAP-43 and MAP-2 mRNA and protein expression in groups B and C at postoperative days 3,7,and 14 showed a trend of first decreasing and then increasing,and the expression in group C was consistently higher than in group B(P<0.05).The expression level of NogoA in group C showed an opposite trend to GAP-43 and MAP-2.Compared with preoperative levels,NogoA mRNA and pro-tein expression in group B rats decreased on postoperative days 1 and 3(P<0.05)and increased on days 7 and 14(P<0.05).Compared with preoperative levels,NogoA mRNA and protein expression in groups B and C also showed a trend of first decreasing and then increasing,but in group C,at all postoperative time points except day 1,it was lower than in group B(P<0.05).Immunofluorescence results showed that over time,the number of cells double-labeled with BrdU and NF-200 gradually increased,with the highest number observed in group C on postoperative day 14.Conclusion NEP1-40 promotes neurological repair in SCI,providing a new approach for SCI repair treatment.

Objective To study the development of conus medullaris by quantitative three-dimensional ultrasound in the second and third trimesters of pregnancy,and to analyze the value of terminal caudal vertebral ossification center count in diagnosis of closed spina bifida.Methods A total of 108 normal fetuses in the second and third trimesters of pregnancy who were admitted to Baoding No.1 Central Hospital from January 2019 to March 2024 were selected and assigned to the normal group.The distance between the fetal conus medullaris and the first sacral vertebra,the distance between the end of the conus medullaris and the end of sacrum,and the number of corresponding vertebrae were measured by three-dimensional ultrasound at different gestational weeks.Meanwhile,54 fetuses who were confirmed to have closed spina bifida after delivery were enrolled into the abnormal group.The number of ossification centers on the median sagittal section of the caudal spine was counted and compared between the two groups.The receiver operating characteristic(ROC)curve was drawn to analyze the value of ossification center count of distal caudal vertebrae in diagnosis of closed spina bifida.Results With the increase of gestational weeks,the distance between the fetal conus medullaris and the first sacral vertebra and the distance between the end of the conus medullaris and the end of the sacral bone were gradually increased.The conus medullaris of normal fetuses was mainly located in L1.5,L2 and L2.5,accounting for 85.19%(92/108).The ossification center count of distal caudal vertebra was slightly increased with the increase of gestational weeks in the normal group.At 17-20 gestational weeks,85.71%(12/14)of the ossification center count of distal caudal vertebra was 5 to 7.At 21-28 gestational weeks,100.00%(45/45)of the ossification center count was 6 and more.At week 29 and above,100.00%(45/45)of the ossification center count was more than 7.In the abnormal group,96.30%(52/54)of the fetuses had the ossification center less than 6,and 85.71%(42/49)of the ossification center count was≤5 at 21-32 gestational weeks.There were significant differences in the ossification center count between the two groups at 17-20,21-24,25-28,29-32,and 33-36 gestational weeks(P<0.05).ROC curve showed that,at 17-20,21-24,25-28,29-32,and 33-36 gestational weeks,the area under the curve(AUC)in prediction of closed spina bifida was 0.804,0.744,0.776,0.819,and 0.722 when the ossification center count of distal caudal vertebra was 5.31,4.59,4.81,4.67,and 5.49 as the cut-off value,respectively.Conclusion The conus medullaris moves upward with the increase of gestational weeks in the second and third trimesters of pregnancy.The ossification center count of the distal caudal vertebra is less than 6 in most fetuses with closed spina bifida,which is conducive to screening for closed spina bifida.

Objective:To clarify the effect of methotrexate on blood uric acid levels and the incidence of hyperuricemia in patients with rheumatic and musculoskeletal diseases (RMDs).Methods:The clinical data were collected from 349 patients with RMDs who took methotrexate for more than 52 weeks and 429 patients with RMDs who did not take methotrexate, who were treated at Anqing Medical Center of Auhui Medical University from June 1, 2022 to June 30, 2024, to compare the differences in serum uric acid concentration and the incidence of hyperuricemia before and after 24 weeks of methotrexate administration in the two groups of patients with RMDs. The changes in serum uric acid concentration and serum creatinine value in the MTX na?ve patients who had taking MTX for 0, 24 and 52 weeks were compared. The relationship between serum uric acid concentration and methotrexate dosage was analyzed. Measurement data were compared using t-test or ANOVA, repeated measures analysis of variance, and count data were compared using χ2 test. Results:①At week 0, there was no significant difference in serum uric acid concentration [(300±63)μmol/L vs. (306±64)μmol/L, t=-1.416, P=0.157] and the incidence of hyperuricemia [9.3%(40/429) vs. 10.3%(36/349) , χ2=0.215, P=0.643] between the two groups. At week24, the serum uric acid concentration (307±70)μmol/L vs. (246±89)μmol/L was statistically significantly ( t=10.909, P<0.001) different. The incidence of hyperuricemia (11.0%, 47/429) vs. (4.6%, 16/349), was statistically significantly different ( χ2=10.497, P<0.001). There was a statistically significant difference in serum uric acid concentration between week 0 and week 24 in the methotrexate group ( t=10.237, P<0.001), and there was a statistically significant difference in the incidence of hyperuricemia ( χ2=8.312, P=0.004). ②The overall serum uric acid concentrations at week 0, weeks 24, and weeks 52 were (306±64)μmol/L, (246±89)μmol/L, and (247±66)μmol/L, respectively. The difference in overall serum uric acid concentration was statistically significant ( F= 29.506, P<0.001). There was no significant difference in serum uric acid concentration between weeks 24 and 52 ( P=1.000). There were significant differences in serum creatinine levels between weeks 0, 24 and 52 ( P<0.001). There was no significant difference in serum creatinine levels between weeks 0 ,52, weeks 24 and 52 ( P=0.077, P=1.000). There were statistically significant differences in the overall serum uric acid concentration and serum creatinine value at weeks 0, 24 and 52 of medication ( P<0.001).③ There was no significant difference in serum uric acid concentration before and after taking hydroxychloroquine, cyclosporine, tripterygium wilfordii, mycophenolate mofetil, tofacitinib, etanercept and adalimumab alone for weeks 0 and 24(all P>0.05). ④There was no significant difference in serum uric acid concentration between patients taking different doses of methotrexate (7.5 mg once weekly, 10 mg once weekly, 12.5 mg once weekly, 15 mg once weekly) at weeks 0 and 24 weeks(all P>0.05). Conclusion:MTX, as an anti-rheumatic drug, reduces the serum uric acid level and the incidence of hyperuricemia in patients with RMDs during the treatment.

BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

Objective:To clarify the effect of methotrexate on blood uric acid levels and the incidence of hyperuricemia in patients with rheumatic and musculoskeletal diseases (RMDs).Methods:The clinical data were collected from 349 patients with RMDs who took methotrexate for more than 52 weeks and 429 patients with RMDs who did not take methotrexate, who were treated at Anqing Medical Center of Auhui Medical University from June 1, 2022 to June 30, 2024, to compare the differences in serum uric acid concentration and the incidence of hyperuricemia before and after 24 weeks of methotrexate administration in the two groups of patients with RMDs. The changes in serum uric acid concentration and serum creatinine value in the MTX na?ve patients who had taking MTX for 0, 24 and 52 weeks were compared. The relationship between serum uric acid concentration and methotrexate dosage was analyzed. Measurement data were compared using t-test or ANOVA, repeated measures analysis of variance, and count data were compared using χ2 test. Results:①At week 0, there was no significant difference in serum uric acid concentration [(300±63)μmol/L vs. (306±64)μmol/L, t=-1.416, P=0.157] and the incidence of hyperuricemia [9.3%(40/429) vs. 10.3%(36/349) , χ2=0.215, P=0.643] between the two groups. At week24, the serum uric acid concentration (307±70)μmol/L vs. (246±89)μmol/L was statistically significantly ( t=10.909, P<0.001) different. The incidence of hyperuricemia (11.0%, 47/429) vs. (4.6%, 16/349), was statistically significantly different ( χ2=10.497, P<0.001). There was a statistically significant difference in serum uric acid concentration between week 0 and week 24 in the methotrexate group ( t=10.237, P<0.001), and there was a statistically significant difference in the incidence of hyperuricemia ( χ2=8.312, P=0.004). ②The overall serum uric acid concentrations at week 0, weeks 24, and weeks 52 were (306±64)μmol/L, (246±89)μmol/L, and (247±66)μmol/L, respectively. The difference in overall serum uric acid concentration was statistically significant ( F= 29.506, P<0.001). There was no significant difference in serum uric acid concentration between weeks 24 and 52 ( P=1.000). There were significant differences in serum creatinine levels between weeks 0, 24 and 52 ( P<0.001). There was no significant difference in serum creatinine levels between weeks 0 ,52, weeks 24 and 52 ( P=0.077, P=1.000). There were statistically significant differences in the overall serum uric acid concentration and serum creatinine value at weeks 0, 24 and 52 of medication ( P<0.001).③ There was no significant difference in serum uric acid concentration before and after taking hydroxychloroquine, cyclosporine, tripterygium wilfordii, mycophenolate mofetil, tofacitinib, etanercept and adalimumab alone for weeks 0 and 24(all P>0.05). ④There was no significant difference in serum uric acid concentration between patients taking different doses of methotrexate (7.5 mg once weekly, 10 mg once weekly, 12.5 mg once weekly, 15 mg once weekly) at weeks 0 and 24 weeks(all P>0.05). Conclusion:MTX, as an anti-rheumatic drug, reduces the serum uric acid level and the incidence of hyperuricemia in patients with RMDs during the treatment.

Objective:To investigate the current situation of the use of transjugular intrahepatic portosystemic shunt (TIPS) for portal hypertension, which should aid the development of TIPS in China.Methods:The China Portal Hypertension Alliance (CHESS) initiated this study that comprehensively investigated the basic situation of TIPS for portal hypertension in China through network research. The survey included the following: the number of surgical cases, main indications, the development of Early-TIPS, TIPS for portal vein cavernous transformation, collateral circulation embolization, intraoperative portal pressure gradient measurement, commonly used stent types, conventional anticoagulation and time, postoperative follow-up, obstacles, and the application of domestic instruments.Results:According to the survey, a total of 13 527 TIPS operations were carried out in 545 hospitals participating in the survey in 2021, and 94.1% of the hospital had the habit of routine follow-up after TIPS. Most hospitals believed that the main indications of TIPS were the control of acute bleeding (42.6%) and the prevention of rebleeding (40.7%). 48.1% of the teams carried out early or priority TIPS, 53.0% of the teams carried out TIPS for the cavernous transformation of the portal vein, and 81.0% chose routine embolization of collateral circulation during operation. Most of them used coils and biological glue as embolic materials, and 78.5% of the team routinely performed intraoperative portal pressure gradient measurements. In selecting TIPS stents, 57.1% of the hospitals woulel choose Viator-specific stents, 57.2% woulel choose conventional anticoagulation after TIPS, and the duration of anticoagulation was between 3-6 months (55.4%). The limitation of TIPS surgery was mainly due to cost (72.3%) and insufficient understanding of doctors in related departments (77.4%). Most teams accepted the domestic instruments used in TIPS (92.7%).Conclusions:This survey shows that TIPS treatment is an essential part of treating portal hypertension in China. The total number of TIPS cases is far from that of patients with portal hypertension. In the future, it is still necessary to popularize TIPS technology and further standardize surgical indications, routine operations, and instrument application.