Sulodexide inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy

Sulodexide is a mixed glycosaminoglycan composed of heparin and dermatan sulfate. In this study, the anti-angiogenic effect of sulodexide was investigated using an oxygen-induced retinopathy (OIR) mouse model. The retinas of sham-injected OIR mice (P17) had a distinctive central area of nonperfusion, and this area was significantly decreased in sulodexide-injected mice. The number of neovascular tufts measured by SWIFT_NV and mean neovascular lumen number were significantly decreased in sulodexide-injected mice. Hyperbaric oxygen exposure resulted in increased levels of VEGF, MMP-2 and MMP-9, and when mice were treated with sulodexide, a dose-dependent reduction in VEGF, MMP-2 and MMP-9 levels was observed. Our results clearly demonstrate the anti-angiogenic effect of sulodexide and highlight sulodexide as a candidate supplementary substance to be used for the treatment of ocular pathologies that involve neovascularization. [BMB Reports 2014; 47(11): 637-642]     

The effect of baicalin in a mouse model of retinopathy of prematurity

Baicalin is a flavonoid derived from the dried root of Scutellaria baicalensis. In this study, oxygen-induced retinopathy was used to characterize the anti-angiogenic properties of baicalin in mice. Pups were exposed to a hyperbaric oxygen environment to induce retinal angiogenesis and were subjected to intraperitoneal injection of baicalin. Avascular area, neovascular tufts, and neovascular lumens were quantified from digital images. Compared to the vehicle, baicalin clearly reduced the central avascular zone and the number of neovascular tufts and lumens. High-dose baicalin (10 mg/kg) significantly reduced the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, angiotensin II, and vascular endothelial growth factor (VEGF). These results show that baicalin is a powerful antiangiogenic compound that attenuates new vessel formation in the retina after systemic administration, and is a candidate substance for therapeutic inhibition of retinal angiogenesis. [BMB Reports 2015; 48(5): 271-276]     

Blockade of TREM-1 prevents vitreoretinal neovascularization in mice with oxygen-induced retinopathy

In pathological retinal neovascularization (RNV) disorders, the retina is infiltrated by activated leukocytes and macrophages. Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, activates monocytes and macrophages and plays an important role in cancer, autoimmune and other inflammation-associated disorders. Hypoxia-inducible TREM-1 is involved in cancer angiogenesis but its role in RNV remains unclear. Here, to close this gap, we evaluated the role of TREM-1 in RNV using a mouse model of oxygen-induced retinopathy (OIR). We found that hypoxia induced overexpression of TREM-1 in the OIR retinas compared to that of the room air group. TREM-1 was observed specifically in areas of pathological RNV, largely colocalizing with macrophage colony-stimulating factor (M-CSF) and CD45- and Iba-1-positive cells. TREM-1 blockade using systemically administered first-in-class ligand-independent TREM-1 inhibitory peptides rationally designed using the signaling chain homooligomerization (SCHOOL) strategy significantly (up to 95%) reduced vitreoretinal neovascularization. The peptides were well-tolerated when formulated into lipopeptide complexes for peptide half-life extension and targeted delivery. TREM-1 inhibition substantially downregulated retinal protein levels of TREM-1 and M-CSF suggesting that TREM-1-dependent suppression of pathological angiogenesis involves M-CSF. Targeting TREM-1 using TREM-1-specific SCHOOL peptide inhibitors represents a novel strategy to treat retinal diseases that are accompanied by neovascularization including retinopathy of prematurity.     

The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.     

两种不同病毒载体携带靶向大鼠金属蛋白酶组织抑制因子（TIMP）-1小干扰RNA抗肝纤维化作用的比较

目的观察以腺相关病毒（AAV）及慢病毒（Lentivirus）为载体含有针对大鼠金属蛋白酶组织抑制因子（TIMP）-1具有较强抑制作用的小干扰RNA（siRNA）对四氯化碳（CCI。）诱导的大鼠肝纤维化模型的干预作用。方法挑选针对大鼠TIMP-1基因具有较强抑制作用的siRNA序列,在体外构建为短发夹表达载体后,将其包装为重组AAV／siRNA-TIMP-I和Lenti／siRNA-TIMP-I,同时包装对照病毒AAV／EGFP和Lenti／EGFP。将Wistar大鼠分为对照组、CCl。模型组、AAV／EGFP组、Lenti／EGFP组、AAV／siRNA-TIMP-I和Lenti／siRNA-TIMP-1组,观察CCl。造模4周后,经H＆E及Masson染色评价各组动物的肝纤维化状况,经荧光定量RCR及Westernblot方法检测TIMP-1的表达抑制情况。结果H＆E及Masson染色证实,与对照组相比,CCI．模型组、AAV／EGFP组、Lenti／EGFP组肝脏胶原纤维明显增加,纤维化评分多为34级,同时肝脏TIMP-1的转录与蛋白表达水平均明显上升;而AAV／siRNA-TIMP-1和Lenti／siRNA-TIMP-l组肝纤维化程度明显减轻,纤维化评分多为2-3级,伴随肝脏TIMP-1的转录与蛋白表达水平均显著抑制。AAV／siRNA-TIMP-l和Lenti／siRNA-TIMP-I组在组织学表现及TIMP-1基因的转录与表达水平上无统计学差异。结论AAV／siRNA-TIMP-I和Lenti／siRNA-TIMP-1均可有效抑制大鼠肝脏TIMP-1基因表达,进而发挥抗肝纤维化作用。     

Dynamics of an HIV-1 therapy model of fighting a virus with another virus

In this paper, we rigorously analyse an ordinary differential equation system that models fighting the HIV-1 virus with a genetically modified virus. We show that when the basic reproduction ratio ?₀<1, then the infection-free equilibrium E ₀ is globally asymptotically stable; when ?₀>1, E ₀ loses its stability and there is the single-infection equilibrium E _s. If ?₀∈(1, 1+δ) where δ is a positive constant explicitly depending on system parameters, then the single-infection equilibrium E _s that is globally asymptotically stable, while when ?₀>1+δ, E _s becomes unstable and the double-infection equilibrium E _d comes into existence. When ?₀ is slightly larger than 1+δ, E _d is stable and it loses its stability via Hopf bifurcation when ?₀ is further increased in some ways. Through a numerical example and by applying a normal form theory, we demonstrate how to determine the bifurcation direction and stability, as well as the estimates of the amplitudes and the periods of the bifurcated periodic solutions. We also perform numerical simulations which agree with the theoretical results. The approaches we use here are a combination of analysis of characteristic equations, fluctuation lemma, Lyapunov function and normal form theory.     

A comparison of the levels of hepatocyte growth factor in serum in patients with type 1 diabetes mellitus with different stages of diabetic retinopathy

INTRODUCTION: The aim of this study was to evaluate the blood concentration of hepatocyte growth factor (HGF) in patients at various stages of retinopathy. We hypothesised that the high level of HGF found in diabetic patients may be an important marker of retinopathy progression and that HGF level may be an index of the risk of proliferative retinopathy. MATERIAL AND METHODS: The participants in the study were 76 patients with type 1 diabetes mellitus. Of these, 35 patients were without retinopathy and formed Group 1. Of the remaining 41 patients with retinopathy, 20 patients had non-proliferative diabetic retinopathy (NPDR) and formed Group 2, while 21 patients had proliferative diabetic retinopathy (PDR) and formed Group 3. We evaluated the concentration of HGF In the peripheral blood by an enzyme-linked immunosorbent assay. RESULTS: Mean serum concentrations of HGF in the control group were significantly lower than in the type 1 diabetic patients. We found a significant increase in HGF serum concentrations in diabetic patients with PDR compared with the control group. Mean serum HGF concentrations were significantly higher in diabetic subjects with PDR than in diabetic patients without retinopathy. CONCLUSION: HGF concentration is increased in patients with type 1 diabetes mellitus with proliferative retinopathy, and concentrations increase with the progression of retinopathy, suggesting that HGF plays a role in the pathogenesis of proliferative diabetic retinopathy.     

出、入肺血中ET-1、VEGF含量与慢性低氧性肺动脉高压的相关性研究

目的：观察间断性减压低氧大鼠出、入肺血血浆、肺组织匀浆和体循环动脉匀浆中内皮素-1（ET-1）的含量变化,以及出、入肺血血清中血管内皮生长因子（VEGF）的含量变化及其与低氧性肺动脉高压的关系。方法：建立间断性减压低氧模型,采用插管法分别测定左侧颈总动脉压力（CAP）、右心室平均压分别代表体循环压力和平均肺动脉压力（mPAP）,放射免疫测定法测定ET-1含量,双抗体夹心ABC—ELISA法测定VEGF含量。结果：①低氧2周组（H2）、低氧3周组（H3）大鼠平均肺动脉压力、右心肥厚指数（RV／LV＋S）均分别显著高于对照组,而各组平均颈动脉压力之间无明显变化（P〉0．05）。②对照组大鼠出肺血中ET含量比入肺血中低（P〈0．05）。H2、H3组大鼠出、入肺血中ET-1含量较对照组均明显增高（P〈0．01）,且H2、H3组大鼠出肺血中ET-1含量均高于入肺血中ET-1含量（P〈0．05）,刚好与对照组相反。H2、H3组大鼠肺组织匀浆中ET—1含量与对照组肺组织和低氧组体循环动脉组织匀浆比较,均明显增高（P〈0.01）,而各组体循环动脉组织匀浆中ET-1含量间无明显差异（P〉0．05）：③对照组大鼠出、入肺血中VEGF含量无明显差异（P〉0．05）,H2、H3组大鼠出肺血血清中VEGF含量较对照组明显增高（P〈0.01）。结论：在慢性低氧可使肺组织产生ET-1和VEGF增多,这可能是慢性肺动脉高压发生、发展的重要机制之一。肺动脉局部缩血管物质ET-1增高,而体循环动脉局部ET-1不增高。可能是长期慢性低氧可导致肺动脉高压的形成,却不发生高血压的机制之一。     

Characterization of T helper (Th)1- and Th2-type immune responses caused by baculovirus-expressed protein derived from the S2 domain of feline infectious peritonitis virus, and exploration of the Th1 and Th2 epitopes in a mouse model

Feline infectious peritonitis virus (FIPV) may cause a lethal infection in cats. Antibody-dependent enhancement (ADE) of FIPV infection has been recognized, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis. In the present study, whether or not the T helper (Th)1 epitope was present in the spike (S)2 domain was investigated, the ADE epitope being thought to be absent from this domain. Three kinds of protein derived from the C-terminal S2 domain of S protein of the FIPV KU-2 strain were developed using a baculovirus expression system. These expressed proteins were the pre-coil region which is the N-terminal side of the putative fusion protein (FP), the region from FP to the heptad repeat (HR)2 (FP-HR2) region, and the inter-helical region which is sandwiched between HR1 and HR2. The ability of three baculovirus-expressed proteins to induce Th1- and Th2-type immune responses was investigated in a mouse model. It was shown that FP-HR2 protein induced marked Th1- and Th2-type immune responses. Furthermore, 30 peptides derived from the FP-HR2 region were synthesized. Five and 16 peptides which included the Th1 and Th2 epitopes, respectively, were identified. Of these, four peptides which included both Th1 and Th2 epitopes were identified. These findings suggest that the identification of Th1 epitopes in the S2 domain of FIPV has important implications in the cat.     

A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF–RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.     

Roles of Kunitz domains in the anti-invasive effect of hepatocyte growth factor activator inhibitor type 1 in human glioblastoma cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine proteinase inhibitor having two extracellular Kunitz-type proteinase inhibitor domains (KD) namely KD-1 and KD-2. It efficiently inhibits hepatocyte growth factor activator, matriptase, hepsin, prostasin and trypsin. We have previously reported that the expression of HAI-1 suppresses the in vitro invasive capability of human glioblastoma cells. In this study we examined the role of each KD in the anti-invasive effect of HAI-1. Engineered over-expression of the mature membrane-form HAI-1 suppressed in vitro fibrin gel invasion of two human glioblastoma cell lines, U251 and YKG-1. The migratory activity on type IV collagen was also suppressed by the HAI-1 expression. These effects were not affected by the deletion of intracytoplasmic domain of HAI-1. A truncated secreted form of HAI-1 also suppressed in vitro invasion of the cells, indicating that the extracellular portion of HAI-1 was responsible for the anti-invasive effect. To determine the roles of each KD in the anti-invasive effect of HAI-1 in vitro, we constructed expression plasmids for HAI-1 with or without mutation at the P1 position of the reactive site of each KD. The results revealed that the proteinase inhibitor activity of N-terminal KD (KD-1) is responsible for the anti-invasion effect of HAI-1.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP

The influenza virus genome replicates in the host cell nucleus, and the progeny viral ribonucleoproteins (vRNPs) are exported to the cytoplasm prior to maturation. The influenza virus NS2 protein has a nuclear export signal (NES) and binds to M1. It is therefore postulated that vRNP is exported from the nucleus by binding to NS2 through M1. However, the significance of the association between NS2 and M1 for the nuclear export of vRNP is still poorly understood. We herein demonstrate that the C-terminal domain of NS2 (residues 81–100) is essential for M1 binding and the nuclear export of progeny vRNPs.

Structured summary

MINT-8057301, MINT-8057317: NS2 (uniprotkb:P03508) binds (MI:0407) to M1 (uniprotkb:P03485) by pull down (MI:0096)     

The expression of vascular endothelial growth factor and its receptors (flt1/fms, flk1/KDR, flt4) and vascular endothelial growth inhibitor in the bovine uterus during the sexual cycle and their correlation with serum sex steroids

The present study was conducted to demonstrate of the immunohistochemical localization of vascular endothelial growth factor (VEGF) and its receptors (flt1/fms, flk1/KDR and flt4) as well as vascular endothelial growth inhibitor (VEGI) and to determine the correlation of VEGF and its receptors and VEGI with serum sex steroids (estrogen and progesterone) in the bovine uterus during the sexual cycle. The stage of the estrous cycle in 30 Holstein cattle was assessed based on the gross and histological appearance of the ovaries and uterus and on blood steroid hormone levels. Tissue samples obtained from the uterus were fixed in 10% formaldehyde for routine histological processing. During both follicular and luteal phases, positive cytoplasmic and membrane staining was achieved for VEGF and its receptors (flt1/fms, flk1/KDR and flt4) as well as VEGI in the luminal and glandular epithelial cells, the connective tissue and smooth muscle cells, and the vascular endothelial cells and smooth muscle cells in the uterus. The intensity, proportional and total scores determined for VEGF and its receptors (flt1/fms and flt4) as well as VEGI were greater in the luminal and glandular epithelial cells compared to the connective tissue and smooth muscle cells (P < 0.05). Furthermore, the number and intensity of the flk1/KDR positive cells were greater among the connective tissue cells compared to the luminal and glandular epithelial cells (P < 0.05). As a result, it was determined that the expression of VEGF and its receptors as well as VEGI in the bovine uterus during the follicular and luteal phases varied with different cell types. This suggests that depending on the stage of the sexual cycle, these factors may mediate the establishment of an appropriate environment for the nutritional supply and implantation of the embryo primarily due to the stimulation of angiogenesis but also through the increase in the secretory activity of the epithelial cells in the uterus. Furthermore, this indicates that ovarian steroid hormones play a significant role in regulating the expression of VEGF and its receptors as well as VEGI.     

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE⁻/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE⁻/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.     

Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B

Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.