Stability of a type 2 diabetes rat model induced by high-fat diet feeding with low-dose streptozotocin injection

Objective

The present study aims at determining the stability of a popular type 2 diabetes rat model induced by a high-fat diet combined with a low-dose streptozotocin injection.

Methods

Wistar rats were fed with a high-fat diet for 8 weeks followed by a one-time injection of 25 or 35 mg/kg streptozotocin to induce type 2 diabetes. Then the diabetic rats were fed with regular diet/high-fat diet for 4 weeks. Changes in biochemical parameters were monitored during the 4 weeks.

Results

All the rats developed more severe dyslipidemia and hepatic dysfunction after streptozotocin injection. The features of 35 mg/kg streptozotocin rats more resembled type 1 diabetes with decreased body weight and blood insulin. Rats with 25 mg/kg streptozotocin followed by normal diet feeding showed normalized blood glucose level and pancreatic structure, indicating that normal diet might help recovery from certain symptoms of type 2 diabetes. In comparison, diabetic rats fed with high-fat diet presented decreased but relatively stable blood glucose level, and this was significantly higher than that of the control group (P>0.05).

Conclusions

This model easily recovers with normal diet feeding. A high-fat diet is suggested as the background diet in future pharmacological studies using this model.

     

Activity of metalloproteinases and adiponectin in obese patients— a possible factor of incisional hernias after bariatric procedures

Purpose

Metalloproteinases are a key component of the pathogenesis of abdominal hernias. Obesity is considered a risk factor in herniogenesis and hernia recurrence. The aim of this study was to evaluate the serum concentrations of metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and adiponectin in morbidly obese and nonoverweight controls.

Materials and methods

The participants were recruited from among patients undergoing bariatric and non-bariatric surgery and divided into two groups: I (body mass index (BMI)≥35 kg/m², n=40) and II (BMI<25 kg/m², n=30). Serum concentrations of MMP-2, MMP-9, MMP-13, and adiponectin were measured using enzyme-linked immunosorbent assay (ELISA).

Results

A statistically significant difference between groups was observed for MMP-2 concentration. The median MMP-9 concentration was higher in the obese group, but the difference was not statistically significant. Median MMP-13 concentrations did not differ between groups. Serum adiponectin concentration was insignificantly higher in the non-obese group.

Conclusions

The elevated serum MMP-2 and MMP-9 concentrations in obese individuals may be related to the higher incidence of incisional hernias in this population.

     

Increasing the immune activity of exosomes: the effect of miRNA-depleted exosome proteins on activating dendritic cell/cytokine-induced killer cells against pancreatic cancer

Background

Tumor-derived exosomes were considered to be potential candidates for tumor vaccines because they are abundant in immune-regulating proteins, whereas tumor exosomal miRNAs may induce immune tolerance, thereby having an opposite immune function.

Objective

This study was designed to separate exosomal protein and depleted exosomal microRNAs (miRNAs), increasing the immune activity of exosomes for activating dendritic cell/cytokine-induced killer cells (DC/CIKs) against pancreatic cancer (PC).

Methods

PC-derived exosomes (PEs) were extracted from cultured PANC-1 cell supernatants and then ruptured; this was followed by ultrafiltered exosome lysates (UELs). DCs were stimulated with lipopolysaccharide (LPS), PE, and UEL, followed by co-culture with CIKs. The anti-tumor effects of DC/CIKs against PC were evaluated by proliferation and killing rates, tumor necrosis factor-α (TNF-α) and perforin secretion. Exosomal miRNAs were depleted after lysis and ultrafiltration, while 128 proteins were retained, including several immune-activating proteins.

Results

UEL-stimulated DC/CIKs showed a higher killing rate than LPS- and PE-stimulated DC/CIKs.

Conclusions

miRNA-depleted exosome proteins may be promising agonists for specifically activating DC/CIKs against PC.

     

<Emphasis Type="Italic">Ureaplasma urealyticum</Emphasis>-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2

Objective

The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipidassociated membrane proteins (LAMPs) in the host innate immune system, specifically their effect on Toll-like receptors (TLRs).

Methods

LAMPs were derived from U. urealyticum strains, and human amniotic epithelial cells (HAECs) were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry.

Results

LAMPs induced HAECs to produce inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor (anti-hTLR2-IgA).

Conclusions

LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.

     

Cognitive-enhancing effects of hydrolysate of polygalasaponin in SAMP8 mice

Objectives

The aim of the study is to evaluate the cognitive-enhancing effects of hydrolysate of polygalasaponin (HPS) on senescence accelerate mouse P8 (SAMP8) mice, an effective Alzheimer’s disease (AD) model, and to research the relevant mechanisms.

Methods

The cognitive-enhancing effects of HPS on SAMP8 mice were assessed using Morris water maze (MWM) and step-through passive avoidance tests. Then N-methyl-d-aspartate (NMDA) receptor subunit expression for both the cortex and hippocampus of mice was observed using Western blotting.

Results

HPS (25 and 50 mg/kg) improved the escape rate and decreased the escape latency and time spent in the target quadrant for the SAMP8 mice in the MWM after oral administration of HPS for 10 d. Moreover, it decreased error times in the passive avoidance tests. Western blotting showed that HPS was able to reverse the levels of NMDAR1 and NMDAR2B expression in the cortex or hippocampus of model mice.

Conclusions

The present study suggested that HPS can improve cognitive deficits in SAMP8 mice, and this mechanism might be associated with NMDA receptor (NMDAR)-related pathways.

     

Hyperbaric oxygen therapy improves the effect of keloid surgery and radiotherapy by reducing the recurrence rate

Objective

Keloids are exuberant cutaneous scars that form due to abnormal growth of fibrous tissue following an injury. The primary aim of this study was to assess the efficacy and mechanism of hyperbaric oxygen therapy (HBOT) to reduce the keloid recurrence rate after surgical excision and radiotherapy.

Methods

(1) A total of 240 patients were randomly divided into two groups. Patients in the HBOT group (O group) received HBOT after surgical excision and radiotherapy. Patients in the other group were treated with only surgical excision and radiotherapy (K group). (2) Scar tissue from recurrent patients was collected after a second operation. Hematoxylin and eosin (H&E) staining was used to observe keloid morphology. Certain inflammatory factors (interleukin-6 (IL-6), hypoxia-inducible factor-1α (HIF-1α), tumor necrosis factor-α (TNF-α), nuclear factor κB (NF-κB), and vascular endothelial growth factor (VEGF)) were measured using immunohistochemical staining.

Results

(1) The recurrence rate of the O group (5.97%) was significantly lower than that of the K group (14.15%), P<0.05. Moreover, patients in the O group reported greater satisfaction than those in the K group (P<0.05). (2) Compared with the recurrent scar tissue of the K group, the expression levels of the inflammatory factors were lower in the recurrent scar tissue of the O group.

Conclusions

Adjunctive HBOT effectively reduces the keloid recurrence rate after surgical excision and radiotherapy by improving the oxygen level of the tissue and alleviating the inflammatory process.

     

Phytochemical composition,biological potential and enzyme inhibition activity of <Emphasis Type="Italic">Scandix pecten-veneris</Emphasis> L.

Objective

Scandix pecten-veneris L. is a less studied wild edible herb and is considered an extinct plant species in many parts of the world. This study was designed to evaluate its phytochemical composition and biological potential of S. pecten-veneris L.

Methods

Phytochemicals including alkaloids, flavonoids, polyphenols, and tannins were determined in extracts of S. pecten-veneris. Antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), while reducing power was tested by ferric reducing/antioxidant power (FRAP) assay. Antimicrobial activity against seven bacterial and four fungal strains was evaluated using agar well diffusion assay. Enzymes inhibition study was performed for urease, phosphodiesterase-I, and catalase-II.

Results

S. pecten-veneris showed moderate antiradical activity and reducing potential of hydroxyl radicals to about 20% of the initial value. The antioxidant activity of various extracts of S. pecten-veneris showed a linear correlation with total phenolic contents in the order of water>n-butanol>chloroform>ethyl acetate>methanol extracts. S. pecten-veneris leaves showed the highest inhibitory activity against Staphylococcus aureus while the highest antifungal activity was observed against Candida albicans. The plant extract was most potent against urease enzymes but showed moderate activity against phosphodiestrase-I and carbonic anhydrase-II.

Conclusions

Our data demonstrate that in addition to its culinary uses, S. pecten-veneris has good medicinal potential and hence could be used for treating some specific health ailments.

     

Enucleation of non-invasive tumors in the proximal pancreas: indications and outcomes compared with standard resections

Objective

The aim of this study was to evaluate the safety and efficiency of enucleation (EU) for proximal pancreatic non-invasive neoplasms.

Methods

Patients with solitary non-invasive neoplasms in the proximal pancreas from January 1998 to April 2014 at the Second Affiliated Hospital of Zhejiang University, Hangzhou, China were included. Different operations and outcomes were analyzed.

Results

A total of 123 patients were enrolled. Forty patients (32.5%) underwent EU including 18 patients who had tumors close to the main pancreatic duct (MPD). Sixty-one patients (49.6%) had pancreaticoduodenectomy (PD) performed and 22 (17.9%) underwent central pancreatectomy (CP). Pathological outcomes included neuroendocrine tumors, cystic lesions, and solid pseudopapillary tumors. Operation time, intra-operative blood loss, and duration of hospital stay were significantly reduced in the EU group. PD was associated with the greatest complication rate (55.7%), followed by EU (50%) and CP (40.9%), though the pancreatic fistula rate after EU was the highest (50%), especially in patients with tumors larger than 3 cm and tumors close to the MPD. EU had advantages in the preservation of pancreatic parenchyma and endocrine and exocrine function.

Conclusions

EU can be carried out safely and effectively for tumors in the proximal pancreas with improved outcomes compared with standard resections, even if the tumor is larger than 3 cm and close to the MPD.

     

Effect of ultraviolet photofunctionalization of dental titanium implants on osseointegration

Objective

The aim of the current study was to evaluate the effect of ultraviolet (UV) photofunctionalization of dental titanium implants with exposure to the oral cavity on osseointegration in an animal model.

Methods

Forty-eight titanium implants (Camlog® Conelog® 4.3 mmx9.0 mm) were placed epicrestally into the edentulous jaws of three minipigs and implant stability was assessed by measuring the implant stability quotient (ISQ). Prior to implantation half of the implants were photofunctionalized with intense UV-light. After three months, the implants were exposed and ISQ was measured again. After six months of implant exposure, the minipigs were sacrificed and the harvested specimens were analyzed using histomorphometric, light, and fluorescence microscopy.

Main results

Forty-two of 48 implants osseointegrated. The overall mean bone-implant contact area (BIC) was (64±22)%. No significant differences were found in BIC or ISQ value (multivariate analysis of variance (MANOVA), P&gt;0.05) between implants with and without exposure to UV photofunctionalization.

Conclusions

No significant effects were observed on osseointegration of dental titanium implants nine months after exposure of UV photofunctionalization.

     

Acupuncture for treating polycystic ovary syndrome: guidance for future randomized controlled trials

Objective

To provide guidance for future randomized controlled trials (RCTs) based on a review concerning acupuncture for treating polycystic ovary syndrome (PCOS).

Methods

A comprehensive literature search was conducted in October 2015 using MEDLINE, EMBASE, SCISEARCH, Cumulative Index to Nursing and Allied Health Literature, the Cochrane Menstrual Disorders and Subfertility Group trials register, Allied and Complementary Medicine (AMED), China National Knowledge Infrastructure (CNKI), and the Wanfang databases. RCTs comparing either acupuncture with no/sham/pharmacological intervention or a combination of acupuncture and conventional therapy with conventional therapy in the treatment of PCOS were included in this review. A quality evaluation was performed for each of the included studies.

Results

Thirty-one RCTs were included in the review and were divided into four categories according to the type of intervention used in the comparator or control group. Menstrual frequency, hormones, anthropometrics, insulin sensitivity, blood lipids, and fertility were used as the main measurements to assess the effects of acupuncture on the patients with PCOS. Thirty trials, except for one, showed an improvement in at least one of the indicators of PCOS after acupuncture treatment. However, normalizing the methodological and reporting format remains an issue.

Conclusions

Based upon this review of current clinical trials concerning acupuncture for treating PCOS, we provide guidelines for better clinical trial design in the future.

     

未受控制的高血糖对2型糖尿病患者粘附分子的影响（英文）

目的:评估可溶性血管细胞间黏附分子(s VCAM-1)、可溶性细胞间黏附分子(s ICAM-1)、可溶性选择素E和可溶性血栓调节蛋白在血糖控制良好和不受控制的2型糖尿病患者中的水平。创新点:对2型糖尿病患者的血管内皮炎症标记物进行评估。方法:62例糖尿病患者分成两组:第一组包括35个血糖控制良好的糖尿病患者,第二组包括27个未控制血糖并伴有微蛋白尿的糖尿病患者。对照组由25名健康志愿者组成。测定血浆中s VCAM-1、s ICAM-1、可溶性选择素E和可溶性血栓调节蛋白的浓度,同时测定血清肌酐及血浆中空腹血糖和糖化血红蛋白(Hb A1c)的浓度。结论:与血糖控制良好的糖尿病组相比,未控制血糖组具有相对低的ICAM-1水平和更高的VCAM-1水平。未控制血糖组中患者的糖化血红蛋白和ICAM-1之间呈正相关,肾小球滤过率和可溶性选择素E之间呈正相关,而肌酐和ICAM-1之间呈负相关。研究证实2型糖尿病的发病机理中炎症过程的出现与血管内皮功能受损有关。未受控制的高血糖对粘附分子的反向作用表明,在糖尿病的并发症中VCAM-1和ICAM-1具有不同功能。     

Comparison of laparoscopic hepatectomy,percutaneous radiofrequency ablation and open hepatectomy in the treatment of small hepatocellular carcinoma

Objective

Three mainstream techniques—laparoscopic hepatectomy (LH), percutaneous radiofrequency ablation (pRFA), and open hepatectomy (OH)—were compared in this study, in terms of their efficacies in the treatment of small hepatocellular carcinoma (HCC).

Methods

A comparative study was performed within a total of 94 patients diagnosed with small HCC in our hospital from 2005 to 2010, who underwent LH (28), RFA (33), or OH (33). They had either a single tumor lesion of less than 5 cm or up to three nodules with diameters of less than 3 cm each. Outcomes were carefully evaluated throughout a 3-year follow-up interval and statistically interpreted.

Results

The pRFA group had a significantly lower disease-free survival rate compared with the two surgical groups (P=0.001) and significantly shorter overall survival (P=0.005), while the LH group and the OH group had no difference in survival results. For patients younger than 60 years old, surgical approaches offered a better long-term overall survival prognosis (P=0.008). There were no statistically significant differences among the three groups in overall survival for elderly patients (P=0.104).

Conclusions

Among patients with small HCC, LH may provide better curative effects than pRFA without increasing complication rates. pRFA leads to faster recurrence than surgical resections. LH has similar therapeutic effects to OH and causes less trauma. For patients younger than 60 years old, LH may be the best curative treatment. Elderly patients may choose either surgery or pRFA.

     

Comparative genomic analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZJ316 reveals its genetic adaptation and potential probiotic profiles

Objective

In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis.

Methods

The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs).

Results

We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis.

Conclusions

Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate.

     

Effects of light curing modes and ethanol-wet bonding on dentin bonding properties

Objective

This study explored the effects of different light curing modes and ethanol-wet bonding on dentin bonding strength and durability.

Methods

A total of 54 molars were randomly divided into three groups: Single Bond 2, Gluma Comfort Bond, and N-Bond. Based on the three light-curing modes and presence or absence of ethanol pretreatment, the samples were assigned to six subgroups: high-light mode, ethanol pretreatment+high-light mode, soft-start mode, ethanol pretreatment+soft-start mode, standard mode, and ethanol pretreatment+standard mode. All samples were bonded with resin based on the experimental groups. After 24 h and 6 months of water storage, a universal testing machine was used to measure microtensile bond strength. Scanning electron microscopy (SEM) was applied to observe mixed layer morphology.

Results

The 24-h and 6-month microtensile bond strengths of the ethanol pretreatment groups were significantly higher than those of the non-ethanol pretreatment groups at the same light modes (P<0.05). With or without ethanol pretreatment, the microtensile bond strengths of the high-light modes were significantly lower than those of the soft-start modes and standard modes (P<0.05). The microtensile bond strengths of samples from the 6-month water storage group significantly decreased compared with those of samples from the 24-h water storage group (P<0.05). The soft-start groups and standard groups formed better mixed layers than the high-light mode groups, whereas the ethanol pretreatment groups formed more uniform mixed layers than those without ethanol pretreatment.

Conclusions

Ethanol-wet bonding technique, soft-start, and standard modes could improve dentin bonding properties.

     

匹伐他汀钙对高胆固醇血症患者动脉粥样硬化的影响（英文）

目的:观察匹伐他汀钙对高胆固醇血症患者外周血管的影响。创新点:首次在国内发现匹伐他汀钙能够改善高胆固醇血症患者肱动脉和颈动脉血管内皮功能而且延缓其动脉粥样硬化发展,并首次证实改善内皮功能是匹伐他汀钙延缓其动脉粥样硬化发展的重要原因。方法:按照入选排除标准,选取本院高胆固醇血症患者(HC),完成超声心动图检查的40例。根据剂量不同,分为两个剂量组:1 mg剂量组20例(男性5例,女性15例,平均年龄(55.20±8.35)岁),2 mg剂量组20例(男性9例,女性11例,平均年龄(57.56±6.09)岁)。访视结束后完成超声心动图检查的HC组36例,两个剂量组分别有2人失访。治疗后1 mg剂量组18例(男性3例,女性15例,平均年龄(56.00±7.85)岁),2 mg剂量组18例(男性7例,女性11例,平均年龄(57.79±6.46)岁)。选择本院同期体检中心30例正常人作为对照(年龄和性别均与病例组匹配,男性14例,女性16例,平均年龄(54.94±6.90)岁)。所有研究对象,均经隔夜禁食12~14小时,次日清晨抽取空腹肘静脉血,测定临床生化指标。采用Sequia512彩色多普勒超声诊断仪,应用高分辩率外周血管超声技术,检测HC治疗前后肱动脉血流介导性舒张功能(FMD)、颈动脉结构和功能。结论:经匹伐他汀钙治疗8周后,高胆固醇血症患者血管功能明显改善,表现为FMD升高,僵硬度减小;颈动脉僵硬度和内中膜厚度(IMT)延缓进展与其内皮功能改善密切相关。     

Complementary feeding patterns among ethnic groups in rural western China

Objective

This study investigated complementary feeding practices among four ethnic groups (Han, Uygur, Tibetan, and Zhuang) based on a cross-sectional survey in rural western China.

Methods

In 2005, a stratified multistage cluster random sampling method was used to recruit 9712 children (7411 Han, 1032 Uygur, 678 Tibetan, and 591 Zhuang) between 6 and 35 months of age and their mothers from 45 counties in 10 provinces (autonomous regions, municipalities) in western China.

Results

The rates of early introduction (before 6 months) of complementary foods in four ethnic groups (Han, Uygur, Tibetan, and Zhuang) were 71.30%, 95.95%, 82.40%, and 72.30%, respectively. The Infant and Child Feeding Index (ICFI) for Uygur and Tibetan children was lower than that for Han children at all age groups. Uygur children were more likely to have unqualified ICFI compared with Han children in a multivariate logistic regression (odds ratio (OR)=5.138, 95% confidence interval (CI): 4.340–6.084). A higher level of maternal education, greater family wealth, and the availability of complementary feeding educational materials decreased the likelihood of an unqualified ICFI. The nutritional status of children (Han, Tibetan, and Zhuang) with qualified ICFI was better than that for children with unqualified ICFI.

Conclusions

Appropriate interventions are required to improve complementary feeding practices in rural western China.

     

Effects of electroacupuncture at 2 and 100 Hz on rat type 2 diabetic neuropathic pain and hyperalgesia-related protein expression in the dorsal root ganglion

Objective

To investigate the analgesic effects of electroacupuncture (EA) at 2 and 100 Hz on type 2 diabetic neuropathic pain (DNP) and on the expressions of the P2X3 receptor and calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG).

Methods

Rat type 2 DNP was induced by a high calorie and high sugar diet fed for 7 weeks, plus a single intraperitoneal injection of streptozotocin (STZ) after 5 weeks. EA at 2 and 100 Hz was carried out once every day after 7 weeks for 7 consecutive days. Body weight, serum fasting insulin (FINS), fasting blood glucose (FBG), insulin sensitivity index (ISI), and paw withdrawal latency (PWL) were measured. The expressions of L4–L6 DRG P2X3 receptors and CGRP were assessed by immunofluorescence.

Results

Type 2 DNP was successfully induced as shown by the increased body weight, FINS, and FBG, as well as the reduced ISI and PWL. Expressions of P2X3 receptors and CGRP in L4–L6 DRGs increased. EA at both 2 and 100 Hz relieved type 2 DNP, but the analgesic effect of EA was stronger at 2 Hz. P2X3 receptor expression decreased in L4–L6 DRGs following EA at 2 Hz and in L5 and L6 DRGs following EA at 100 Hz. EA at both 2 and 100 Hz down-regulated CGRP overexpression in L4–L6 DRGs.

Conclusions

These findings indicate that EA at 2 Hz is a good option for the management of type 2 DNP. The EA effect may be related to its down-regulation of the overexpressions of the DRG P2X3 receptors and CGRP in this condition.

     

Vitamin D status among infants,children, and adolescents in southeastern China

Objective

Vitamin D deficiency and insufficiency are global public health problems, which must first be identified before they can be appropriately addressed, and yet information is strikingly lacking in most parts of the Asia and Pacific region. The study aimed to document and account for the actual situation in Wenzhou on the southeastern coast of China.

Subjects and methods

Serum 25-hydroxyvitamin D levels among a total of 5845 infants, preschool children, school children, and adolescents aged 1–18 years were examined between March 2014 and February 2015.

Results

Their mean levels were (110.2±26.8), (77.5±25.7), (55.6±15.4), and (47.2±13.9) nmol/L, respectively. Older age groups were involved in increasing risk of vitamin D deficiency and insufficiency. There were significant seasonal differences in its median level and prevalence of deficiency and insufficiency among school children and adolescents, but there was no significant sex difference in mean level and prevalence in any age group.

Conclusions

Vitamin D deficiency and insufficiency were prevalent among infants, preschool children, school children, and adolescents in Wenzhou. A vitamin D-rich diet and outdoor activities for 1–2 h per day under the natural conditions favorable to its endogeous synthesis do not suffice. The vitamin D status in Wenzhounese infants excelling over that in the US was the result of its supplementation thanks to the Chinese Medical Association recommendations, which should be consequently extended to more age groups. Life style shaped by socio-economic environments affects vitamin D status. Knowledge on the importance of vitamin D for healthy growth should be popularized.