Serum-plasma matched metabolomics for comprehensive characterization of benign thyroid nodule and papillary thyroid carcinoma.

Metabolomics offers a noninvasive methodology to identify metabolic markers for pathogenesis and diagnosis of diseases. This work aimed to characterize circulating metabolic signatures of benign thyroid nodule (BTN) and papillary thyroid carcinoma (PTC) via serum-plasma matched metabolomics. A cohort of 1,540 serum-plasma matched samples and 114 tissues were obtained from healthy volunteers, BTN and PTC patients enrolled from 6 independent centers. Untargeted metabolomics was determined by liquid chromatography-quadrupole time-of-flight mass spectrometric and multivariate statistical analyses. The use of serum-plasma matched samples afforded a broad-scope detection of 1,570 metabolic features. Metabolic phenotypes revealed significant pattern differences for healthy versus BTN and healthy versus PTC. Perturbed metabolic pathways related mainly to amino acid and lipid metabolism. It is worth noting that, BTN and PTC showed no significant differences but rather overlap in circulating metabolic signatures, and this observation was replicated in all study centers. For differential diagnosis of healthy versus thyroid nodules (BTN + PTC), a panel of 6 metabolic markers, namely myo-inositol, α-N-phenylacetyl-L-glutamine, proline betaine, L-glutamic acid, LysoPC(18:0) and LysoPC(18:1) provided area under the curve of 97.68% in the discovery phase and predictive accuracies of 84.78-98.18% in the 4 validation centers. Taken together, serum-plasma matched metabolomics showed significant differences in circulating metabolites for healthy versus nodules but not for BTN versus PTC. Our results highlight the true metabolic nature of thyroid nodules, and potentially decrease overtreatment that exposes patients to unnecessary risks.

Rapid preparation of auto-healing gels with actuating behaviour.

Gels with multiple stimuli-responsive actuating behaviour have shown great potential in many applications. Nevertheless, facile approaches to rapidly preparing gel actuators are still highly needed, and obtaining gels possessing both actuating and auto-healing capabilities remains a challenge. Herein, we report the rapid preparation of gel actuators with a self-healing ability. Dual-component gels, composed of poly(BA-co-VI-co-AM) (G-1) and poly(BA-co-AA-co-AM/ß-CD) (G-2) (BA = butyl acrylate, VI = N-vinyl imidazole, AM = acrylamide, AA = acrylic acid, ß-CD = ß-cyclodextrin), are prepared within 10 minutes (min) via biphase frontal polymerization (FP). Both G-1 and G-2 gels show excellent intrinsic self-healing properties based on hydrogen bonds, with healing efficiencies of 91% and 97%, respectively; self-healing between G-1 and G-2 also occurs due to hydrogen bonding and host-guest interactions. Moreover, dual-component gels, in terms of G-1 and G-2 bilayer gel flowers and strips, heterogeneous healed bilayer gel strips, and microfluidic-directed bilayer gel microsphere ensembles, all show actuating behaviour in acidic, alkaline and organic solutions, with actuation degrees up to 96% in 5 min. The actuation mechanism is also proposed. This work might provide new insights into fast synthesis of self-healing dual-component gels towards application in the actuator field.

[Clinical characteristics of paraneoplastic retinopathy and optic neuropathy].

OBJECTIVE: To analyze the clinical characteristics of paraneoplastic retinopathy and optic neuropathy(PRON). METHODS: Case series study. Eight patients were enrolled from October 2006 to March 2012 visited in ophthalmology department, the People Liberation Army General Hospital. The patients were underwent a series of examinations, including fundus photography, visual electrophysiology, fundus fluorescein angiography, optic coherent tomography,fundus autofluorescent imaging, perimetry, ultrasonography, magnetic resonance imaging, spinal tap and cerebrospinal fluid test, paraneoplastic syndrome (PNS) antibody test. The patients were followed up in outpatient department and(or) by phone. The clinical manifestation,entity types, and treatment were analyzed. RESULTS: Of the eight patients, there were cancer associated retinopathy(CAR) 3 cases, bilateral diffuse uveal melanocytic proliferation (BDUMP) 2 cases and paraneoplastic optic neuropathy(PON) 3 cases. Five patients were detected the PNS antibodies and revealed three patients with positive results. As for the primary malignancy,four of the eight patients were lung carcinoma,others included invasive thymoma, kidney cancer, acute lymphocytic leukemia and cervical cancer, each for one case. All the patients complained vision blurring or progressive visual decrease. Other complaints included dark shadow in two patients, shimmering, dazzling, double vision and eye pain, each in one patient. One patient complained progressive decreased vision in both eyes prior to the diagnosis of lung cancer. Of the 16 eyes of the eight patients, there were six patients with no light perception vision, five from light perception to 0.05, and other five with no less than 0.4 vision, in the end of the follow up. Five patients were treated with steroid with unsatisfactory efficacy. CONCLUSIONS: Each entity of PRON has its own clinical characteristics. PRON especially BDUMP may be a pre-metastatic disease.

Comparison of outcomes of idiopathic macular holes treated by vitrectomy with air or silicone oil tamponade based on the hole size.

AIM: To compare the efficacy of vitrectomy combined with air or silicone oil in the treatment of idiopathic macular hole (IMH). METHODS: According to the results of high-definition optical coherence tomography (HD-OCT), 75 cases (75 eyes) of IMH in stage II-IV (Gass stage) in the General Hospital of Chinese PLA from January 2017 to December 2019 were collected for this retrospective study. The best corrected visual acuity (BCVA) and minimum diameter of IMH (MMHD) were measured. Eyes underwent vitrectomy combined with internal limiting membrane peeling operation, and were divided into disinfection air group (30 eyes) and silicone oil group (45 eyes) according to the intraocular tamponade. For MMHD≤400 µm (MMHD1), there were 23 eyes in air group and 16 eyes in silicone oil group. For MMHD2>400 µm (MMHD2), there were 7 eyes in air group and 29 eyes in silicone oil group. One month after surgery, the closure rates of IMH and BCVA were compared and analyzed. According to HD-OCT, the closure shape was graded with A (bridge closure) and B (good closure). RESULTS: The closure rates of air group and silicone oil group were 86.67% and 95.56% respectively with no significant difference (P>0.05); For MMHD1, those of air group and silicone oil group were 95.65% and 100% respectively with no significant difference (P>0.05); For MMHD2, those of air group and silicone oil group were 57.14% and 93.10% respectively, and those of the silicone oil group were higher than the air group (P<0.05). There was no significant difference in the closure shape grade between MMHD1 air group and silicone oil group (P>0.05). The proportion of Grade B in MMHD2 silicone oil group was higher than that in the air group (P<0.05). BCVA of each group after operation was better than that before operation, and there was no significant difference between air group and silicone oil group. While among them, MMHD1 air group was better than silicone oil group (P<0.05), and there was no significant difference between MMHD2 air group and silicone oil group (P>0.05). CONCLUSION: For smaller IMH (≤400 µm), the efficacy of vitrectomy combined with air should be considered better than silicone oil; for larger IMH (>400 µm), the efficacy of silicone oil may be better than air.

Untargeted metabolomics and lipidomics uncovering the cardioprotective effects of Huanglian Jiedu Decoction on pathological cardiac hypertrophy and remodeling.

ETHNOPHARMACOLOGICAL RELEVANCE: As a classic herbal prescription, Huanglian Jiedu Decoction (HLJDD) exhibits positive effects against cardiac dysfunction. However, its cardioprotective effects and potential mechanism(s) of action still need to be systematically investigated. AIM OF THE STUDY: This study aimed to reveal the underlying therapeutic mechanism of HLJDD on transverse aortic constriction (TAC)-induced pathological cardiac hypertrophy and remodeling. MATERIALS AND METHODS: TAC-induced cardiac hypertrophy and remodeling mice model was established to evaluate the therapeutic effects of HLJDD. Serum untargeted metabolomics and lipidomic profiling were performed using ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry coupled with multivariate statistical analyses. RESULTS: Oral administration of HLJDD (2.5 g/kg/day, 5.0 g/kg/day) significantly improved the heart morphology, enhanced the heart function, and alleviated the accumulation of fibrosis in the interstitial space and the infiltration of inflammatory cells in TAC-stimulated mice. Serum untargeted metabolomics analysis showed that significant alterations were observed in metabolic signatures between the TAC-model and sham group. Principal component analysis and orthogonal partial least-squares discriminant analysis screened 59 differential metabolic features and 13 metabolites were identified. The disturbed metabolic pathways in TAC group mainly related to lipid metabolism. Further serum lipidomic profiling showed that most lipids including cholesterol esters, ceramides, glycerides, fatty acids and phospholipids were decreased in TAC group and these alterations were reversed after HLJDD intervention. CONCLUSION: HLJDD alleviates TAC-induced pathological cardiac hypertrophy and remodeling, and its potential therapeutic mechanism involves the regulation of lipid metabolism.

Cerebrospinal fluid-based metabolomics to characterize different types of brain tumors.

BACKGROUND: Brain tumors cause significant morbidity and mortality due to rapid progression and high recurrence risks. Reliable biomarkers to improve diagnosis thereof are desirable. OBJECTIVE: This work aimed to identify panels of biomarkers for diagnostic purposes using cerebrospinal fluid (CSF)-based metabolomics. METHODS: A cohort of 163 histologically-proven patients with brain disorders was involved. Comprehensive CSF-based metabolomics was achieved by liquid chromatography-quadrupole time-of-flight spectrometric (LC-Q/TOF-MS) and multivariate statistical analyses. The diagnostic performance of the metabolic markers was evaluated using receiver operating characteristic curves. RESULTS: A total of 508 ion features were detected by the LC-Q/TOF-MS analysis, of which 27 metabolites were selected as diagnostic markers to discriminate different brain tumor types. The area under the curve (AUC) was 0.91 for lung adenocarcinoma patients with brain metastases (MBT) vs. lung adenocarcinoma patients without brain metastases (NMBT), 0.83 for primary central nervous system lymphoma (PCNSL) vs. secondary central nervous system involvement of systemic lymphoma (SCNSL), 0.77 for PCNSL vs. MBT, 0.87 for SCNSL vs. MBT, 0.86 for MBT vs. nontumorous brain diseases (NT), and 0.80 for PCNSL vs. NT. Perturbed metabolic pathways between the comparisons related mainly to amino acids and citrate metabolism. CONCLUSIONS: CSF-based metabolomics to a large extent reliably identifies significant metabolic differences between different brain tumors and shows great potential for diagnosis of brain tumors.

IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART.

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

Three new C-27-carboxylated-lupane-triterpenoid derivatives from Potentilla discolor Bunge and their in vitro antitumor activities.

Three new lupane-triterpenoids (1-3) along with six known compounds (4-9) were isolated from the ethanolic extract of whole plant of Potentilla discolor Bunge. The structures of Compounds 1-3 were established by extensive 1D and 2D NMR together with other spectrum analysis, indicating that their C-27 positions were highly oxygenated, which were rarely found in nature. Their in vitro anti-proliferative activities against HepG-2, MCF-7 and T-84 cell lines were evaluated by Cell Counting Kit-8 (CCK-8) assay, and the results showed different activities for three cell lines with IC50 values ranging from 17.84 to 40.64 µM. In addition, the results from Hoechst 33258 and AO/EB staining as well as annexinV-FITC assays exhibited Compound 1 caused a markedly increased HepG-2 cellular apoptosis in a dose-dependent manner. The further mechanisms of Compound 1-induced cellular apoptosis were confirmed that 1 induced the production of ROS and the alteration of pro- and anti-apoptotic proteins, which led to the dysfunction of mitochondria and activation of caspase-9 and caspase-3 and finally caused cellular apoptosis. These results would be useful in search for new potential antitumor agents and for developing semisynthetic lupane-triterpenoid derivatives with high antitumor activity.

[The dynamic study of retinoic acid and its alginate sodium microspheres in rabbit eye].

OBJECTIVE: To obtain a intravitreous delay release system by injection of biodegradable polymers (alginate sodium-retinoic acid microspheres, AGS-RA microspheres) to the rabbit vitreous and study the release character of RA delivery system in vitro. METHODS: RA was mixed with 1.5% alginate sodium and microspheres was made by a special electrostatic generator after dissolved in organic solvent; The content of AGS-RA in the microspheres and its release from the microspheres were measured by spectrophotometer. The characteristic of the metabolic dynamics of AGS-RA in the microspheres was analyzed by HPLC. RESULTS: The size of microspheres we manufacture is (95.2443 +/- 8.6265) microm; Medicine contain quantity of RA is (1.7644 +/- 0.0453) microg/mg; Vitro test show that RA in the microspheres is release evenly in the 28 days of observation; Inject medicine to vitreous cavity show no poisonous side effect; The medicine dynamics research of microspheres showed that medicine is being even in 6 weeks releasing [the aqueous RA density of 1, 3 d, 1, 2, 3, 4, 5, 6 weeks is (23.79 +/- 0.15), (33.45 +/- 0.48), (19.95 +/- 0.79), (21.12 +/- 0.47), (19.65 +/- 0.35), (20.01 +/- 0.25), (18.24 +/- 0.27), (18.5 +/- 0.68) ng/ml, respectively]. Just in the 3rd day there is a release summit. CONCLUSION: In vivo and in vitro tests show that AGS-RA microspheres can delay RA release uniformity.

Peripheral blood Dendritic cells are not a major reservoir for HIV type 1 in infected individuals on virally suppressive HAART.

Dendritic cells (DCs) are potent antigen-presenting cells, and their physiological localization in tissues that interact with the external environment is important as a first barrier against pathogens such as human immunodeficiency virus type I (HIV-1). Several models have been proposed to explain the possible role of DCs as a reservoir for HIV-1 in patients on virally suppressive highly active antiretroviral therapy (HAART). However, the low yield of cell isolates has made this evaluation a difficult task. The present study analyzes whether peripheral blood DCs from HIV-1-infected individuals on virally suppressive HAART, with plasma HIV-1 RNA levels of less than 50 copies/ml, carry either HIV-1 provirus and/or HIV-1 virions. Peripheral blood DCs were isolated from a cohort of 10 HIV-1-seropositive men taking suppressive HAART. In five patients, plasmacytoid and myeloid dendritic cells were isolated to attempt to identify their respective roles in HIV-1 residual disease. Viral out-growth assays were performed in vitro, as well as gag and R/U5 polymerase chain reaction (PCR) amplification of viral RNA and DNA, respectively, from DC and peripheral blood mononuclear cell (PBMC) extracts. Fluorescence activated cell-sorting (FACS) data revealed cellular yields from 85.90 to 95.18%, of relatively pure DCs isolated from patients' PBMCs. Although HIV-1 RNA gag and DNA RU/5 were detected in all PBMC samples isolated from the patients, proviral DNA and viral RNA forms were not detected in any of the DC isolates. In addition, no replication-competent virus was demonstrated in DC coculture assays, while virus was isolated from each patients' CD8+ T-lymphocyte-depleted PBMC cocultures. Furthermore, HIV-1 gag proviral DNA was not detected in either plasmacytoid or myeloid DC subfractions. The current study suggests that in HIV-1-infected individuals treated with suppressive HAART, peripheral blood DCs do not carry HIV-1 proviral DNA or viral particles attached to their surface. These populations of peripheral blood DCs are likely not a major HIV-1 reservoir in patients on HAART with clinically undetectable plasma viral RNA.

[Clinical application of keratoprosthesis for corneal opacity unsuitable for keratoplasty].

OBJECTIVE: To evaluate the results of keratoprosthesis for complicated corneal opacities unsuitable for keratoplasty. METHODS: Fifteen keratoprosthesis were implanted in 15 patients with bilateral blindness. Preoperative visual acuity (VA) was light perception in all operated eyes. The corneal opacities were caused by severe alkali burn (6 eyes), sulfate acid injury (3 eyes), melting aluminum injury (2 eyes), explosive injury (1 eye), severe dry eye (1 eye) and multi-keratoplasty failure (2 eyes). The keratoprosthesis (MICOF) were made by Moscow eye microsurgery complex in Russia. Surgical techniques consisted of two stages; first, inserting a supporting titanium frame into the lamellar pocket and then, implanting an optical part 3 months later. RESULTS: Follow-up time ranged from 4 to 26 months (9.5 months on average). In 14 of 15 eyes (93%), postoperative VA ranged from 0.12 to 1.0 without correction. Corrected postoperative VA was: 7 eyes (47%) with VA from 0.6 to 1.2; 5 eyes (33%) from 0.3 to 0.5 and 2 eyes (13%) from 0.15 to 0.2. The reason of the only eye without VA improvement was a pre-existing retinal detachment. CONCLUSION: Keratoprosthesis improves vision for corneal opacity patients with complicated conditions such as dense neovascularization and severe ocular surface disorders.

Influence of plasma and cerebrospinal fluid levels of endothelin-1 and NO in reducing cerebral vasospasm after subarachnoid hemorrhage during treatment with mild hypothermia, in a dog model.

Using vascular heat-exchange controller implemented mild hypothermia treatment, the authors established the cerebral vasospasm model in which blood was injected twice into dog's foramen magnum; and it was discussed the influence of the concentration of endothelin-1 and NO in blood plasma and cerebrospinal fluid through continuing treatment of mild hypothermia at different times in secondary brain vasospasm model after subarachnoid hemorrhage. Thirty healthy mongrel dogs were randomly divided into five groups; artificial cerebrospinal fluid group (group A), normal temperature control group (group C), mild hypothermia 8 h group (group H1), mild hypothermia 16 h group (group H2), and mild hypothermia 32 h group (group H3). The authors injected the artificial CSF into dog's foramen magnum in group A while the other four groups were injected with autologous arterial blood. The normal group's temperature maintained 38.5°C. The authors set the temperature at 33.5°C in mild hypothermia groups and this was maintained for 8, 16, and 32 h, respectively. ET-1 and NO levels in the cerebrospinal fluid and plasma were assayed in each group on days 0, 7, 14, and 21. Then the changes of the diameter of blood vessels of cerebral basilar artery and overall performance categories score in each group through application of CT angiography were recorded. In the cerebral vasospasm model which was constructed by injecting the blood to dog twice, mild hypothermia treatment, through the application of vascular heat-exchange controller, could reduce cerebral vasospasm. It was observed that the duration of the mild hypothermia is directly proportional to the longer duration of the relieving of cerebral vasospasm. The reciprocal changes observed in the levels of ET-1 and NO in cerebrospinal fluid and plasma revealed that it might be possible to reduce the cerebral vasospasm by regulating the rising amplitude of ET-1 and the decrease in NO in CSF and plasma.

APOBEC3G upregulation by alpha interferon restricts human immunodeficiency virus type 1 infection in human peripheral plasmacytoid dendritic cells.

APOBEC3G (A3G), a member of cytidine deaminase family, has potent anti-human immunodeficiency virus type 1 (HIV-1) activity. It has been demonstrated that alpha interferon (IFN-alpha) can significantly enhance the expression of A3G in human primary resting CD4(+) T-cells, macrophages and primary hepatocytes, subsequently decreasing their viral susceptibility. Plasmacytoid dendritic cells (pDCs) are key effectors in innate host immunity, mediating adaptive immune responses and stimulating IFN-alpha production in reaction to various stimuli. In this report, we demonstrate that IFN-alpha, either exogenously added to- or endogenously secreted by pDCs, can enhance the expression of A3G and its family members such as A3A, A3C and A3F. We have also shown that IFN-alpha can inhibit HIV-1 expression in pDCs. This inhibitory effect could be countered by addition of an A3G-specific short interfering RNA, indicating that IFN-alpha-induced A3G plays a key role in mediating pDCs response to HIV-1. Given the central role played by pDCs in orchestrating the IFN-alpha/A3G intercellular network and intracellular signal pathway, our data indicate that pDCs themselves are also protected by an IFN-alpha/A3G-mediated innate immunity barrier from HIV-1 infection.

Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells.

The interferon (IFN) system, including various IFNs and IFN-inducible gene products, is well known for its potent innate immunity against wide-range viruses. Recently, a family of cytidine deaminases, functioning as another innate immunity against retroviral infection, has been identified. However, its regulation remains largely unknown. In this report, we demonstrate that through a regular IFN-alpha/beta signal transduction pathway, IFN-alpha can significantly enhance the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) in human primary resting but not activated CD4 T cells and the amounts of APOBEC3G associated with a low molecular mass. Interestingly, short-time treatments of newly infected resting CD4 T cells with IFN-alpha will significantly inactivate human immunodeficiency virus type 1 (HIV-1) at its early stage. This inhibition can be counteracted by APOBEC3G-specific short interfering RNA, indicating that IFN-alpha-induced APOBEC3G plays a key role in mediating this anti-HIV-1 process. Our data suggest that APOBEC3G is also a member of the IFN system, at least in resting CD4 T cells. Given that the IFN-alpha/APOBEC3G pathway has potent anti-HIV-1 capability in resting CD4 T cells, augmentation of this innate immunity barrier could prevent residual HIV-1 replication in its native reservoir in the post-highly active antiretroviral therapy era.

Emergence of autologous neutralization-resistant variants from preexisting human immunodeficiency virus (HIV) quasi species during virus rebound in HIV type 1-infected patients undergoing highly active antiretroviral therapy.

The role of neutralizing antibodies (NAbs) during virus rebound in human immunodeficiency virus type 1 (HIV-1)-infected patients undergoing highly active antiretroviral therapy is poorly understood. Three patients in this study had NAbs to preexisting autologous HIV-1 and an episode of virus rebound after a prolonged period of virus suppression. To investigate the influence of NAbs on virus evolution, envelope genotypes of preexisting and rebound viruses were examined. Phylogenetic analysis of env (V1-V5) sequences indicated that rebound viruses had evolved from or preexisted in baseline populations. By use of envelope pseudotype viruses, rebound viruses were found to be significantly resistant to neutralization by autologous antibody in all 3 patients, indicating that rebound viruses were selected by NAbs. The site responsible for conferring neutralization resistance against autologous antibody was identified in the upstream C3 region in 2 of 3 patients.