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Objective:To discuss the differential effects of apolipoprotein E(APOE)gene polymorphism in the neurotoxicity-reactive astrocytes,and to provide the theoretical basis for the study of the pathogenesis of Alzheimer's disease(AD).Methods:The primary cortical astrocytes from the APOE-knockout mice(APOE-/-)were isolated and cultured in vitro,and the purity of the cells was identified by immunofluorescence staining.The human APOE3 and APOE4 recombinant over-expression plasmids were constructed and separately transfected into the primary APOE-/-astrocytes,and the APOE-/-primary cells were regarded as control.Western blotting method was used to detect the expression levels of APOE and glial fibrillary acidic protein(GFAP)proteins in the cells;enzyme-linked immunosorbent assay(ELISA)method was used to detect the APOE level in the cellular culture supernatant.The inflammatory models were prepared with the primary astrocytes transfected with APOE3 and APOE4 and co-stimulated with interleukin-1α(IL-1α),tumor necrosis factor(TNF),and complement C1q.The cells were divided into APOE3+PBS group,APOE4+PBS group,APOE3+IL-1α+TNF+ C1q group,and APOE4+IL-1α+TNF+C1q group.Cell immunofluorescence staining method was used to observe the morphology of the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of glypican 4(Gpc4),glypican 6(Gpc6),thrombospondin 1(Thbs1),thrombospondin 2(Thbs2),SPARC-like protein 1(Sparcl1)and glial cell line derived neurotrophic factor(GDNF),C3,and S100 calcium binding protein B(S100B)mRNA in the cells in various groups;microsphere phagocytosis assay was used to detect the phagocytic capacities of the cells in various groups;Western blotting was used to detect the protein expression levels of B-cell lymphoma 2(Bcl-2),and cysteinyl aspartate specific protease-3(Caspase-3)proteins in the cells in various groups.Results:Compared with APOE-/-group,the expression levels of APOE and GFAP proteins in the cells and the APOE level in the cellular culture supernatant in transfected APOE3 and transfected APOE4 groups were increased(P<0.01).The fluorescence microscope observation results showed that compared with APOE3+PBS and APOE4+PBS groups,the astrocytic processes in APOE3+IL-1α +TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group became shorter and the cell bodies became larger;compared with APOE3+IL-1α +TNF+Cq1 group,the astrocytic processes in APOE4+IL-1α +TNF+Cq1 group were even shorter.Compared with APOE3+PBS and APOE4+PBS groups,the expression levels of Gpc4,Gpc6,Thbs1,Thbs2,and Sparcl1 mRNA in the cells in APOE3+IL-1α +TNF+Cq1 group and APOE4+IL-1α +TNF+Cq1 group were significantly decreased(P<0.01);compared with APOE3+IL-1α +TNF+Cq1 group,the expression levels of Gpc4,Gpc6,Thbs1,Thbs2,and Sparcl1 mRNA in the cells in APOE4+IL-1α +TNF+Cq1 group were significantly decreased(P<0.05 or P<0.01).Compared with APOE3+PBS and APOE4+PBS groups,the expression levels of GDNF mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+ IL-1α +TNF+Cq1 group were decreased(P<0.01),and the expression levels of C3 and S100B mRNA were increased(P<0.01);compared with APOE3+IL-1α +TNF+Cq1 group,the expression level of GDNF mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased(P<0.05),and the expression levels of C3 and S100B mRNA were increased(P<0.05).Compared with APOE3+ PBS group and APOE4+PBS group,the numbers of hagocytosis of microspheres in the cells in APOE3+ IL-1α +TNF+Cq1 group and APOE4+IL-1α +TNF+Cq1 group were significantly decreased;compared with APOE3+IL-1α+TNF+Cq1 group,the number of hagocytosis of microspheres in the cells in APOE4+IL-1α+TNF+Cq1 group was significantly decreased.Compared with APOE3+PBS group and APOE4+PBS group,the expression levels of Bcl-2 protein in the cells in APOE3+IL-1α+TNF+ Cq1 group and APOE4+IL-1α +TNF+Cq1 group were decreased(P<0.05 or P<0.01)and the expression levels of Caspase-3 protein were significantly increased(P<0.01);compared with APOE3+ IL-1α+TNF+Cq1 group,the expression level of Bcl-2 protein in the cells in APOE4+IL-1α+TNF+ Cq1 group was decreased(P<0.01),and the expression level of Caspase-3 protein was increased(P<0.05).Conclusion:The APOE4 genotype has a stronger ability to induce the inflammatory factors compared with APOE3;it can lead to a neurotoxicity-reactive astrocyte phenotype,increase the neurotoxicity,affect the astrocyte apoptosis,and aggravate the neuron damage.

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Objective:To report the clinical manifestation and genetic characteristics of a case of de novo Huntington′s disease due to paternal intermediate alleles. Methods:Clinical data and imaging features of a middle-aged female, who complained of unstable walking without positive family history and was admitted to Xuanwu Hospital, Capital Medical University on September 20, 2022, were retrospectively analyzed. The serum samples of the patient and her parents were used to screen HTT gene dynamic mutation in accordance with the principle of informed consent and voluntary. And the relevant literatures were reviewed. Results:This is a 38-year-old female with progressive course, who presented as ataxia, involuntary movement at the end of extremities, dystonia, and cognitive impairment. Imaging results showed atrophy of bilateral caudate nuclei, as well as decreased glucose metabolism of bilateral caudate nuclei, putamen and partial cortex. Genetic testing showed the abnormal expansion of polymorphic trinucleotide (CAG) repeats in HTT gene and confirmed the diagnosis of Huntington′s disease. The CAG repeat length of the patient was 17/47 (pathopoiesis), of the father was 17/35 (intermediate alleles), and of the mother was 17/17 (normal). Conclusions:Paternal intermediate alleles may cause the first case of Huntington′s disease in a family. Importantly, HTT gene screening should be performed for the patient and parents when the diagnosis of Huntington′s disease is clinically possible despite negative family history, to prevent the misdiagnosis.

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Objective To investigate the correlation between plasma inflammatory factors [IL-1β, IL-6, IL-10, IL-12, IL-17, IL-23, TNF-α, TGF-β, IFN-γ, C-reactive protein (CPR) CCL-5] and hand-foot syndrome in colorectal cancer patients after taking capecitabine. Methods 35 colorectal cancer patients treated with capecitabine were collected and the degree of severity was divided according to the hand-foot syndrome grading diagnostic criteria. The concentrations of inflammatory factors in plasma were determined by ELISA kits. Results The standard curve of all inflammatory cytokines were linear (r>0.9900), and plasma concentrations of inflammatory cytokines in patients with colorectal cancer were determined. The concentration of TNF-α changed obviously, which had reference value. Conclusion The concentrations of different inflammatory factors were different and the concentration of TNF-α was closely correlated with the severity of hand-foot syndrome.

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OBJECTIVE@#To develop a novel cognitive screening tool for older adults in China.@*METHODS@#"Game-based Cognitive Assessment-3 Minute Version"(G3) was designed and developed based on WeChat mini-program. And its feasibility was analyzed.@*RESULTS@#G3 mini-program contains three one-minute mini digital games and supports users' self-assessment of cognitive functions with instant access to reports. G3 had a good correlation with Montreal Cognitive Assessment Basic (MoCA-B) with Pearson's r =0.611 (P<0.001). Among natural users aged 50 and older (71 179), the G3 initiation and completion rates were 99.55% and 92.28%, respectively. The average time to complete G3 assessments was (278.5±73.73) seconds.@*CONCLUSIONS@#The novel G3 mini-program has good feasibility and usability for older Chinese adults, and can be used for cognitive screening and home self-assessment.

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As endogenous chemical substances,neurotransmitters play a vital role in maintaining normal life activities.Abnormal levels of neurotransmitters can lead to physical,mental,and some neurodegenerative diseases.However,the ultralow concentration,complex chemical properties,and release modes of neurotransmitters make their accurate detection in vivo a great challenge.To accurately monitor neurotransmitters in the brain and accurately understand the release kinetics of neurotransmitters,we reviewed several method commonly used in the past five years to detect neurotransmitters in vivo and their research progress.The basic principle and applicability of microdialysis,electrochemical sensors,and fluorescence sensors are introduced in detail.

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The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); host cell entry by this virus relies on the interaction between the receptor-binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor on cell membranes. In addition to serving as a receptor for SARS-CoV-2, ACE2 was originally discovered as a protective factor in the renin-angiotensin system (RAS) that catalyses the degradation of angiotensin II (Ang II) to Ang 1-7, which is involved in multiple organ pathology. Recent genetic and clinical studies reported that ApoE4 expression is associated with increased susceptibility to SARS-CoV-2 infection and the development of severe COVID-19, but the underlying mechanism is currently unclear. In the present study, by using immunofluorescence staining, molecular dynamics simulations, proximity ligation assay (PLA) and coimmunoprecipitation (Co-IP) combined with a biolayer interferometry (BLI) assay, we found that ApoE interacts with both the spike protein and ACE2 but does not show obvious isoform-dependent binding effects. These data suggest that ApoE4 increases SARS-CoV-2 infectivity in a manner that may not depend on differential interactions with the spike protein or ACE2. Importantly, further immunoblotting and immunofluorescence staining results showed that ApoE4 significantly downregulates ACE2 protein expression in vitro and in vivo and subsequently decreases the conversion of Ang II to Ang 1-7, which could worsen tissue lesions; these findings provide a possible explain by which ApoE4 exacerbates COVID-19 disease.

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Objective:To explore the value of magnetic resonance imaging (MRI) parameters combined with serum macrophage migration inhibitory factor (MIF) and microRNA-1203 (miR-1203) in the early diagnosis of hepatocellular carcinoma (HCC) and its relationship with efficacy.Methods:From October 2017 to October 2019, 100 patients with HCC in Hunan Provincial Hospital of Traditional Chinese Medicine were selected as the observation group. 92 patients with benign liver tumor and 102 healthy people were randomly selected as the control group and normal control group. The clinicopathological features, MRI parameters [hepatic artery perfusion index (HPI), volume transfer constant (K trans)], serum MIF and miR-1203 levels were compared among the three groups; the value of MRI parameters, serum MIF and miR-1203 in single and combined diagnosis of HCC was explored; the relationship between each index and curative effect of HCC patients was analyzed. Results:The levels of HPI, K trans, serum MIF and miR-1203 in observation group were higher than those in control group and normal control group ( P<0.05); There were significant differences in HPI, K trans, serum MIF and miR-1203 levels among patients with different tumor length, differentiation degree, Child Pugh grade and distant metastasis in the observation group ( P<0.05). Among HPI, K trans and serum MIF, miR-1203, the specificity of HPI and K trans for diagnosis of HCC were the largest (94.57%), and the sensitivity of K trans for diagnosis of HCC was the largest (75.00%); the area under the curve (AUC) of the combined diagnosis of HCC was 0.879, which was greater than the AUC of the single diagnosis of HPI, K trans and serum MIF, miR-1203 (0.753, 0.793, 0.792, 0.809); the optimal sensitivity and specificity of the combined diagnosis were 79.00% and 86.96%, respectively; the levels of HPI, K trans, serum MIF, and miR-1203 in effective patients in the observation group were lower than those in the ineffective patients after treatment ( P<0.05); the clinical efficacy of HCC patients were significantly correlated with the levels of HPI, K trans, serum MIF and miR-1203 ( P<0.05). Conclusions:The MRI parameters HPI, K trans and serum MIF, miR-1203 levels in patients with HCC increased significantly, which has high application value in assisting clinical diagnosis of HCC, and is closely related to the clinical efficacy of patients, and has great development potential in efficacy evaluation.

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Objective To establish a method to determine 11 main components in Hanshi yufei decoction. Methods The method adopted UHPLC-MS/MS with an Agilent ZORBAX SB-C18 (3.5 μm，2.1 mm×150 mm) column. The mobile phase was consisted of 0.2% formic acid plus 10 mmol/L ammonium acetate aqueous solution(A) - acetonitrile(B) and gradient elution (0–0.6 min, 80%–40%A; 0.6–1 min, 40%–30%A; 1–4.3 min, 30%–5%A) at 0.3 ml/min. The column temperature was 40 ℃ and 11 main components including vanillic acid, magnolol, honokiol, wogonin, sophorin, 6-gingerol, citrinin, qianghuo alcohol, nobiletin, nodakenin, and hesperidin were quantified in a multiple reaction monitoring mode. The reserpine was the standard. Results The 11 main components in Hanshi Yufei decoction had a good linear relationship within their concentration range (r>0.98), and the average recovery was 93.11%～111.73%. Conclusion The UHPLC-MS/MS method established in this experiment is easy to operate and has good reproducibility, which provides a laboratory basis for the quality control of Hanshi Yufei decoction.

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Samples of 88Rb were produced by the irradiation of U3O8. A series of procedures were applied to extract pure radioactive solution of 88Rb. Experimental data was recorded by a 4πßγ-coincidence measurement system. Two rounds of experiments were performed to obtain four sets of measurement data. The measured half-life of 88Rb obtained from the average of the 4πß and 4πßγ-coincidence methods was 17.78 ± 0.05 min, in good agreement with previously reported values but with a significantly reduced uncertainty.

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Radiation-induced brain injury (RBI) is the most serious complication of head and neck tumor after radiotherapy. The pathogenesis of RBI is complicated, and the clinical course is irreversible, while no effective treatment available. The activation of glial cells is one of the main theories of RBI, and the prevention and treatment of RBI by targeting glial cells is the focus of current research. As a post-transcriptional regulatory factor, microRNA (miRNA) has been confirmed to be involved in regulatingglial cell radiosensitivity, inflammation type transformation, autophagy, exosomatic, long non-coding RNA (lncRNA), circular RNA (circRNA) and other related pathways, thereby mediating the occurrence and development of cascade reaction of inflammatory injury and neurological function repair of central nervous system (CNS) disease. Therefore, the establishment of miRNA - glial regulatory network may provide a new strategy for the prevention and treatment of RBI.

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Objective To investigate the extraction methods for active components from oral ulcer film and optimize the determination methods of active components dexamethasone sodium phosphate and metronazole. Methods Different extraction solvents(methanol, water and 70% methanol aqueous) were applied to extract the active components dexamethasone sodium phosphate and metronazole from oral ulcer film, which contents were quantified by a HPLC method. Results the extraction solvent water had the best efficacy and more simpler compared to the other two solvents. Clotriazole showed a good linear relationship within 5.014 5-200.5800 μg/ml (r=0.999 8), and the average extraction recovery was (104.23±0.63)%, and for dexamethasone sodium phosphate, a good linear relationship was obtained in the range of 0.482-16.328 μg/ml (r=0.9999), and the average extraction recovery was (103.97±1.02)%. Conclusion The water extraction method established in this study was simple and efficient, which showed features of simplicity, accuracy and repeatable.

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Alzheimer disease (AD) is a common neurodegenerative disorder in the elderly. It is the most common cause of dementia and is difficult to diagnose in the early stage. PET imaging has important value for early diagnosis, and amyloid imaging is the best choice for early diagnosis of AD, glucose imaging has great value of assessment of disease condition. The application and progresses of PET imaging in early diagnosis of AD, focusing on glucose imaging, amyloid protein imaging and tau protein imaging were reviewed in this article.

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Objective Objective TO investigate the potential association between matrix metalloproteinase-9 (MMP-9) gene polymorphisms and risk of ischemic stroke in Western Guangdong population.Methods This hospitalbased case-control study recruited 251 patients with ischemic stroke and 96 controls.Using Multiplex SNaPshot method was used to detect the genotype of MMP-9 gene rs3787268、rs3918241 and rs3918242 polymorphisms.The association between MMP-9 gene polymorphisms and risk of ischemic stroke was analyzed.Results ① There were significant differences in the genotype distribution of rs3787268 between ischemic stroke group and the controls (P=0.042).In the recessive model,the individual risk of A/A genotype was higher (OR=2.21,P=0.046) than that of the G/G+G/A genotype.② Compared with the controls,the genotype and allele distribution of rs3918242 in the ischemic stroke group were significantly different (P=0.007,P=0.038).In the dominant model,the risk of individuals carrying the T genotype was significantly elevated (OR=2.14,P=0.009) compared with individuals with the C/C genotype.③ The genotype distribution of rs3787268 polymorphisms in the LAA but not in no-LAA subgroup was significantly different from that in the controls (P =0.039).The genotype distribution and allele frequency of rs3918242 polymorphisms in the LAA subgroup were significantly different from that of the control group (P=0.009,P=0.047).There was no significant difference in the genotype distribution and allele frequency between no-LAA subtype and the control group.Conclusions The MMP-9 gene rs3918242 and rs3787268 polymorphisms may be the risk factors of ischemic stroke in Han population in the western part of Guangdong province,China.The MMP-9 gene rs3918242 and rs3787268 polymorphisms may be the risk factors of large-artery atherosclerotic stroke.

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Objective To investigate the effects of substrate stiffness on the proliferation of human umbilical vein endothelial cells ( HUVEC) during dengue virus infection.Methods Polyacrylamide gels were prepared for cell culture [(0±4) kPa].The proliferation of HUVEC cultured on substrates with differ-ent stiffness was determined by using 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-etrazolium,inner salt ( MTS) assay.The cycle and apoptosis of HUVEC were determined by flow cytometry analysis.Dengue virus serotype 2 (DENV-2) strains were propagated and identified by con-ventional assays.The HUVEC were infected with DENV-2 strains at a MOI of 10 and cultured on traditional plastic and hydrogel substrates, respectively.The levels of nitric oxide (NO) and endothelin-1 (ET-1) were detected by nitric acid reductase assay and double antibody sandwich ELISA.Results Young′s modulus E value of the hydrogels was (3030 ±0.44) Pa.The proliferation of HUVEC and the expression of NO and ET-1 were enhanced along the increased substrate stiffness.However, no significant differences with the cell cycle and apoptosis were observed between cells cultured on different substrates.Conclusion The stiffness of substrates affected not only the proliferation of HUVEC, but also the release of cytokines during DENV-2 infection.The development of dengue fever was associated with the decreased secretion of vascular active substances as a result of blood vessel injury.The establishment of hydrogel substrates as the model of vascu-lar basement membranes might provide a new way for the in vitro investigation of the pathogenesis of DENV infection.

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Objective To analyze the effects of dengue virus 2 ( DENV-2 ) infection on the ex-pression of IL-29 in primary human umbilical vein endothelial cells ( HUVECs) cultured on hydrogel sub-strates .Methods Primary HUVECs were isolated and cultured on hydrogel substrates .DENV-2 stains were used to infect the primary HUVECs at a multiplicity of infection( MOI) of 10.Flow cytometry analysis was performed to detect the apoptosis and infection rate of HUVECs after 48 hours of culturing .The gene chip profiling was performed to analyze mRNA expression .The expression of IL-29 at mRNA and protein levels were measured by real-time fluorescent quantitative PCR analysis and double antibody sandwich ELISA as -say, respectively.Results Compared with 96.36%of baby hamster kidney (BHK) cells that were infected with DENV-2 stains, only 4.71%primary HUVECs cultured on hydrogel substrates were infected .The pri-mary HUVECs cultured on hydrogel substrates with or without DENV-2 infection showed no significant differ-ences with the rates of cell apoptosis and infection (P>0.05).A significant difference was observed with the expression of IL-29 at mRNA level between primary HUVECs cultured on hydrogel substrates and the cells cultured in plastic bottles (P<0.05).The results of the real-time quantitative PCR analysis and ELISA as-say showed that IL-29 was highly expressed in DENV-2 infected primary HUVECs cultured on hydrogel sub-strates as compared with those in control groups (P<0.05).Conclusion The expression of IL-29 was de-tected in DENV-2 infected primary HUVECs cultured on hydrogel substrates , which was significantly differ-ent from that in DENV-2 infected primary HUVECs cultured in plastic bottles .The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investi -gation of the pathogenesis of DENV infection .

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Objective To study and optimize the preparation process and abserve the related properties of electret flu-orouracil gel patch .Methods the adhesives and moistening agents were screened by preliminary experiment .The optimal matrix proportion in the blank gel patch was obtained with orthogonal design .The charge storage stability of the patch was studied by compensation method .The transdermal behaviors of the patch were evaluated by modified diffusion cell method and HPLC .Results The optimal matrix proportions of PAAS ∶ gelatin ∶ PVA ∶ Kaolin ∶ CMC-Na ∶ moistening agents were 0 .2∶0 .7∶0 .5∶0 .7∶0 .2∶12 .The patch had a good charge storage stability .All the electrets and 3% azone could enhance the cumulative penetration amount of drug and they had synergistic enhancing effect .Conclusion The qualified electret fluorouracil gel patch was prepared under the optimal process ,which had no skin irritation and the behavior of transdermal de-livery was better than that of the traditional gel patch .

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Objective To prepare and optimize insulin-loaded N-trimethyl chitosan nanoparticles via orthogonal design , and preliminarily study the effects on blood glucose level .Methods The insulin-loaded N-trimethyl chitosan nanoparticles (INS-NPs) were prepared by ionic gelation ,and the optimal proportion was obtained with orthogonal design .The nanoparticles were characterized by transmission electron microscopy and zeta/sizer nano analyzer .The entrapment efficiency of insulin-load-ed nanoparticles and the in vitro releasing characteristics were studied by HPLC .A preliminary pharmacodynamic study of the insulin-loaded nanoparticles was also carried out .Results The INS-NPs exhibited narrow distribution .The mean diameter of INS-NPs was (63 .26 ± 1 .88)nm ,while the entrapment efficiency was (37 .92 ± 2 .11)% .After 8 hours of subcutaneous injec-tion of the INS-NPs ,the blood glucose level in diabetic rats decreased to normal level and kept stable .Conclusion The opti-mized insulin-loaded nanoparticles had homogeneous spherical shape ,small size ,and could be a new approach of administration for insulin .

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High mobility group box 1 (HMGB 1 ) is a family member of the high mobility group. HMGB1 binds to cell surface specific receptor-receptor for advanced glycation end products (RAGE) and toll-like receptor 2 (TLR2), and thus it exhibits extensive biological activities. HMGB1 participates in pathophysiological processes including inflammatory response and regulation of blood-brain barrier permeability in central nervous system, arid it is closely correlated with the diseases such as ischemia stroke, Alzheimer's disease and gliocytoma.

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OBJECTIVE:To study the effect of oil phase,emulsifier and drugs on reverse microemulsion.METHODS:Pseudo-ternary phase diagram was adopted to study the effect of Span-80/Tween-80(emulsifier),thymopentin(model drug), long-chain glyceride,medium-chain glyceride,non-glyceride(oil phase).on the preparation of reverse microemulsions.Optimal formula of reverse microemulsions was selected.RESULTS:The largest water-in-oil(W/O)area was obtained in reverse microemulsion which was prepared using medium-chain glyceride as oil phase.Final formula was confirmed and contained distilled water/ Span-80/Tween-80/caprylic/capric triglyceride(2∶3∶6∶9).In addition,thymopentin was incorporated into the aqueous phase.CONCLUSIONS:The preparation of reverse microemulsions will be influenced by emulsifier,constitution of oil phase and drug.The influence factors for the actual preparation of reverse microemulsions should be optimized.

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In this study, the effect of glycerol-induced acute renal failure (ARF) on the pharmacokinetics of lidocaine after transdermal application was investigated in rats. Microdialysis method was applied in vitro and in vivo to the abdominal skin of rats. After topical application of 1% lidocaine, the cumulative amount of lidocaine permeated through the excised rat abdominal skin showed parallel effect between normal and ARF rats with no significant difference in the in vitro permeability coefficient of lidocaine between them, while area under the plasma concentration versus time curve of lidocaine in ARF rats increased significantly. The protein binding rate of lidocaine in ARF plasma and the blood vessel permeability to muscle tissues, assessed by beta-D-glucopyranosyl fluorescein isothiocyanate-labeled (FITC) albumin, increased significantly. After intravenous infusion of 5 mg/h/kg lidocaine, both of the total body clearance and the volume of distribution of lidocaine in the ARF rats decreased significantly. These results suggested that renal dysfunction did not have any effect on the skin permeability of lidocaine, but might change the plasma protein binding of drug and blood vessel permeability which led to high plasma concentration of lidocaine.

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