Salvage peroral endoscopic myotomy is a promising treatment for achalasia after myotomy failure.

BACKGROUND AND AIMS: Reintervention modalities after myotomy failure in achalasia patients have yet to be established. The efficacy and safety of salvage peroral endoscopic myotomy (POEM) for treatment of achalasia after myotomy failure were evaluated in the study. METHODS: Between August 2011 and August 2021 at the Endoscopy Center of Zhongshan Hospital, 219 achalasia patients who had previously undergone a myotomy underwent a salvage POEM and were thus retrospectively enrolled in this study. After propensity score matching (PSM), operation-related parameters were compared between the salvage POEM group and the naïve POEM group. Subgroup analysis was performed between patients with previous Heller myotomy (HM) and patients with previous POEM. RESULTS: With similar baseline characteristics between both groups after PSM, the salvage POEM group presented with shorter tunnel length (11.8 ± 2.2 cm vs 12.8 ± .9 cm, P < .0001) and myotomy length (9.8 ± 2.0 cm vs 10.4 ± 1.0 cm, P < .0001) than the naïve POEM group. No significant differences were found in procedure-related adverse events between patients of salvage POEM and naïve POEM. The primary outcome of treatment success occurred in 175 of 193 patients (90.7%) in the salvage POEM group versus 362 of 374 patients (96.8%) in the naïve POEM group (P = .0046). At a 2- and 5-year follow-up, significantly higher rates of clinical failures were observed in the previous HM subgroup than in the previous POEM subgroup (P = .0433 and P = .0230, respectively). CONCLUSIONS: Salvage POEM after a previous myotomy failure, especially after a POEM failure, is a promising treatment option because it has a durable clinical relief rate.

Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering.

The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes. Specific insertion sites in the vectors showed significant differences in the expression of fluorescence proteins. Selected integration sites were applied in the de novo synthesis of salicylic acid, leading to a titer of 142.0 ± 6.0 mg/L in a shake flask with good production stability. Additionally, the design successfully realized the biosynthesis of ergothioneine (45 mg/L) by one-step construction. This work expands the application scope of native cryptic plasmids to the easy construction of functional pathways. KEY POINTS: • Cryptic plasmids of EcN were designed to express exogenous genes • Insertion sites with different expression intensities in cryptic plasmids were provided • Target products were stably produced by engineering cryptic plasmids.

Repeat endoscopic submucosal dissection as salvage treatment for local recurrence of esophageal squamous cell carcinoma after initial endoscopic submucosal dissection.

BACKGROUND AND AIMS: Local recurrence of esophageal squamous cell carcinoma (ESCC) after endoscopic resection does not have an established treatment. The efficacy and safety of repeat endoscopic submucosal dissection (ESD) for recurrent ESCC were determined in the study. METHODS: Forty-three consecutive patients with 45 locally recurrent superficial ESCC lesions undergoing repeat ESD and 909 first ESD lesions for propensity score matching (PSM) at Zhongshan Hospital between January 2011 and January 2020 were retrospectively enrolled. After PSM (1:2), operation-related parameters were compared between repeat ESD and first ESD. In the repeat ESD group, the Kaplan-Meier method and log-rank tests were used for identification of risk factors for local recurrence after repeat ESD. RESULTS: As compared with propensity score-matched first ESD, rates of complete resection (86.7% vs 97.8%, P = .02) and curative resection (86.7% vs 96.7%, P = .06) were lower and procedure duration (54.8 ± 21.7 minutes vs 46.2 ± 20.6 minutes, P = .67) and hospital stay (4.3 ± 1.8 days vs 2.9 ± 1.4 days, P = .25) were longer in the repeat ESD group. The en-bloc resection rate (93.3% vs 98.8%, P > .11) remained comparable. Adverse events including bleeding (4.4% vs 0%, P = .11), perforation (.0% vs .0%, P > .99), and stricture (6.7% vs 2.2%, P = .33) presented with no difference. The 5-year overall survival rate and recurrence-free survival rate for repeat ESD was 100% and 86.0%, respectively. Multiplicity was significantly associated with recurrence after repeat ESD (P = .01). CONCLUSIONS: Repeat esophageal ESD showed favorable short- and long-term outcomes and thus provides an alternative choice for recurrent superficial ESCC.

Endoscopic radial incision versus endoscopic balloon dilation as initial treatments of benign esophageal anastomotic stricture.

BACKGROUND AND AIM: We aim to evaluate the efficacy and safety of endoscopic radial incision (ERI) versus endoscopic balloon dilation (EBD) treatment of naïve, recurrent, and refractory benign esophageal anastomotic strictures. METHODS: One hundred and one ERI, 145 EBD, and 42 ERI combined with EBD sessions were performed in 136 consecutive patients with benign esophageal anastomotic stricture after esophagectomy at Zhongshan Hospital from January 2016 to August 2021. Baseline characteristics, operational procedures, and clinical outcomes data were retrospectively evaluated. Parameters and recurrence-free survival (RFS) were compared between ERI and EBD in patients with naïve or recurrent or refractory strictures. Risk factors for re-stricture after ERI were identified using univariate and multivariate analyses. RESULTS: Twenty-nine ERI versus 68 EBD sessions were performed for naïve stricture, 26 ERI versus 60 EBD for recurrent strictures, and 46 ERI versus 17 EBD for refractory stricture. With comparable baseline characteristics, RFS was greater in the ERI than the EBD group for naïve strictures (P = 0.0449). The ERI group had a lower 12-month re-stricture rate than the EBD group (37.9% vs 61.8%, P = 0.0309) and a more prolonged patency time (181.5 ± 263.1 vs 74.5 ± 82.0, P = 0.0233). Between the two interventions, recurrent and refractory strictures had similar RFS (P = 0.0598; P = 0.7668). Multivariate analysis revealed initial ERI treatment was an independent predictive factor for lower re-stricture risk after ERI intervention (odds ratio = 0.047, P = 0.001). Few adverse events were observed after ERI or EBD (3.0% vs 2.1%, P = 0.6918). CONCLUSIONS: ERI is associated with lower re-stricture rates with better patency and RFS compared with EBD for naive strictures.

A highly selective probe for ratiometric imaging peroxynitrite in living cells and in vivo.

In this study, we constructed and displayed a ratiometric fluorescent probe JQ-2 for detecting ONOO-. The probe JQ-2 showed a ratiometric signal for visualizing ONOO- with a rapid response and high selectivity over a panel of biological analytes. Moreover, the JQ-2 has near-infrared emission (657 nm), which provides an excellent basis for the practical application in biological systems. The probe JQ-2 possessed low cytotoxicity and excellent cell membrane permeability, which can specifically visualize the exogenous and endogenous ONOO- in vitro and vivo by emission in two channels. Meanwhile, JQ-2 can be used for diagnosing drug-induced liver injury by visualizing and monitoring the fluctuations of endogenous ONOO-. Therefore, JQ-2 provided a potential tool for precisely detecting the fluctuation of ONOO- in biological systems to understand physiological and pathological process.

Landscape of esophageal submucosal tunneling endoscopic resection-related adverse events in a standardized lexicon: a large volume of 1701 cases.

BACKGROUND: Submucosal tunneling endoscopic resection (STER) has been widely applied for esophageal submucosal tumors. This large volume study aims to provide a standard landscape of STER-related AEs for reference. METHODS: 1701 patients with esophageal SMTs undergoing STER were included at Zhongshan Hospital, Fudan University. Data of clinical characteristics and adverse events were collected and analyzed in depth. Adverse events were recorded by ASGE lexicon and graded by ASGE grading/Clavien-Dindo system. Risk factors for major AEs were analyzed by univariate and multivariate logistic regression. RESULTS: Three hundred and twenty (18.8%) patients with 962 cases of adverse events were observed. Accordingly, 84 (5.0%) were classified as major AEs (moderate and severe) by ASGE grading and 37 (2.2%) were classified as major AEs (grades III-V) by Clavien-Dindo grading. First 1 year operation, distance > 6 cm from incision to tumor, piecemeal resection, partially extraluminal location, mucosal injury, and operation time > 60 min were included in the risk score model for major AEs of STER, with 57.1% sensitivity and 87.5% specificity. CONCLUSIONS: STER was a safe procedure for diagnosis and treatment of esophageal SMTs with a total 18.8% incidence of AEs, among which only 5.0% were major AEs requiring therapeutic measurements.

Calorie restriction conferred improvement effect on long-term rehabilitation of ischemic stroke via gut microbiota.

Calorie restriction can modulate the gut microbiota and protect against many diseases including ischemic stroke. However, the role of calorie-restriction-induced microbiota alteration remained unknown in ischemic stroke rehabilitation. Here we conducted 30% reduction of caloric intake on mice for four weeks, to evaluate its role on ischemic stroke rehabilitation. Significantly, this calorie restriction led to better long-term rehabilitation in comparison of normal control. Notably, the transplantation of gut microbiome from calorie-restriction-treated mice to post-stroke mice was eligible to obtain better long-term rehabilitation of stroke mice. Bifidobacterium identified by 16 S ribosomal RNA sequencing were enriched in those of calorie-restriction mice. Then we administrated Bifidobacterium to stroke mice and found Bifidobacterium treatment could successfully improve the long-term rehabilitation of cerebral ischemia mice. Furthermore, the metabolomics analysis revealed a panel of upshifting metabolites, suggesting that calorie restriction greatly altered the gut microbiota composition and its metabolism. Hence, we discovered the novel effect of CR on long-term rehabilitation of ischemic stroke and the underlying role of gut microbiota, which might provide novel thoughts for the clinical post-stroke rehabilitation.

Incidence of de novo autoimmune hepatitis in children and adolescents with increased autoantibodies after liver transplantation: a meta-analysis.

The objective of this meta-analysis was to assess the incidence of de novo autoimmune hepatitis (AIH) in children and adolescents with increased autoantibodies after liver transplantation. We systematically retrieved studies from PubMed, Embase, Central, CNKI, VIP and Wanfang published before February 1, 2020. All analyses were conducted using R-4.0.1 statistical package (Meta). Seven studies with high quality were pooled in our final analysis (N = 251 participants). The incidence of de novo AIH was 9% [95% confidence interval (CI) 1-23%, I2  = 86%]. Subgroup analysis suggested that publications not using the International Autoimmune Hepatitis Group (IAIHG) criteria have marginally significantly higher incidence of de novo AIH than those using IAIHG criteria (P for interaction = 0.08). The incidence of chronic rejection was 8% (95% CI 2-17%, I2  = 72%). Meta-regression indicated significant correlation (P = 0.04; estimate: 1.51) between the incidence of de novo AIH and the rate of increase of antibodies to liver/kidney microsome (anti-LKM). It is still challenging to distinguish de novo AIH and chronic rejection in children and adolescents with increased autoantibodies after liver transplantation. The diagnostic criteria for de novo AIH in children and adolescents and the role of anti-LKM in the development of de novo AIH deserve future investigation.

Short- and long-term outcomes of endoscopic submucosal dissection for superficial esophageal squamous cell cancer in patients with prior gastrectomy.

BACKGROUND: The surgery for esophageal cancer arising after prior gastrectomy is technically difficult with high morbidity and mortality. Endoscopic submucosal dissection (ESD) is a minimally invasive endoscopic treatment for superficial SCC with high curative resection rate. But few studies are concerned about ESD under these circumstances. The aim of this study was to elucidate the short- and long-term outcomes of ESD for superficial esophageal squamous cell cancer (SCC) in patients with prior gastrectomy. METHODS: From January 2009 to January 2019, 37 patients with prior gastrectomy who underwent ESD for superficial esophageal SCC were retrospectively enrolled at the Zhongshan Hospital, Fudan University in Shanghai, China. Rates of en bloc resection, complete resection, curative resection, incidence of postoperative bleeding, perforation and postoperative stricture were evaluated as short-term outcomes. Overall survival, and local recurrence-free survival were evaluated as long-term outcomes. RESULTS: The rate of en bloc resection, complete resection and curative resection were 94.6%, 86.5% and 78.4%, respectively. No perforation was observed. 1 (2.7%) patient experienced postoperative bleeding. During the median observation of 43 months, 3 (8.6%) patients experienced esophageal stricture, successfully managed by balloon dilation. 3(8.6%) patients had local recurrence after ESD with 5-year local recurrence-free survival rate of 91.4%. During the observation period, 4 patients died of other reasons. The 1, 3, 5-year overall survival rates were 97.1%, 97.1% and 91.4%. CONCLUSIONS: The short-term outcomes indicate ESD is technically difficult with lower resection completeness in patients after gastrectomy, while the long-term outcomes are rather favorable.

Exposure to artificial wind increases energy intake and reproductive performance of female Swiss mice (Mus musculus) in hot temperatures.

High temperatures and heatwaves are rapidly emerging as an important threat to many aspects of physiology and behavior in females during lactation. The body's capacity to dissipate heat is reduced by high ambient temperatures, increasing the risk of hyperthermia. Exposure to wind, a pervasive environmental factor for most terrestrial animals, is known to increase heat loss, but its effects on the reproductive performance of small mammals remains unclear. In the present study, the effects of wind on the energy budgets, resting metabolic rate and milk energy output (MEO) were measured in lactating Swiss mice at 21 and 32.5°C. Females kept at 32.5°C had a significantly lower resting metabolic rate, food intake and MEO, and lighter offspring, than those kept at 21°C. However, exposure to wind increased the asymptotic food intake of females kept at 32.5°C by 22.5% (P<0.01), their MEO by 20.7% (P<0.05) and their litter mass by 17.6% (P<0.05). The body temperature of females kept at 32.5°C was significantly higher during lactation than that of females kept at 21°C, but this difference was reduced by exposure to wind. These findings suggest that exposure to wind considerably improves reproductive performance, increasing the fitness of small mammals while undergoing hot temperatures during heatwaves.

The energy budget and fat accumulation in striped hamsters (Cricetulus barabensis) during post-lactation.

Adaptive adjustments of energy intake and body fat play an important role in allowing animals' to meet the energy demands of thermoregulation during cold conditions and reproduction. Body fat is usually metabolized during lactation, which is one of the most energetically demanding activities of female mammals, however the effect of this on the energy budget and body fat regulation after lactation remains unclear. We compared the energy intake and body fat of female striped hamsters (Cricetulus barabensis) fed either a high-fat or low-fat diet for 21 days after the end of lactation (post-lactation, PL) to those of virgin controls. Serum leptin levels and the expression of hypothalamic orexigenic and anorexigenic neuropeptide genes were also measured and compared. Although lactating females consumed significantly more food, they had significantly lower body fat than virgin controls. The energy intake and body fat levels of the PL females were, however, significantly higher than those of virgin females. This was particularly true for the PL females that were fed high-fat diet. These females had significantly higher serum leptin concentrations, but lower hypothalamic leptin receptor gene expression, than virgin females. Neither orexigenic nor anorexigenic neuropeptide levels in the hypothalamus differed significantly between the PL and virgin females. This suggests that a negative energy balance during lactation drives fat accumulation after lactation. Furthermore, leptin resistance may occur after the end of lactation, causing females to consume more food, and accumulate more fat, than virgin females.

[Roles of Cilia in Heart Development and Congenital Heart Disease].

Cilia are protruding cell structures on the cell surface and are found in almost every type of cell.According to the different structures and quantity of tubulins,cilia can be divided into two categories:motor cilia and sensory cilia.Sensory cilia are also called non-motor cilia and primary cilia,due to the composition and number of tubulins.They are closely related to the development of internal organs and many human physiological activities.Recent studies have demonstrated that cilia are involved in regulating the formation of left and right symmetry of the heart structure,and eventually the heart develops into the left-right asymmetry structures.Since congenital heart diseases(CHD)are characterized by abnormalities in the spatial structure of the heart chamber and outflow tract,cilia may play an important role in the pathogenesis of CHD.Cilia,mainly through ciliary transduction signal pathways,regulate both the formation of left and right asymmetrical structures and the polarity and the migration of cells.Therefore,a clear understanding of the regulation mechanism of ciliary signaling pathway on heart development can provide new therapeutic targets and new ideas for the clinical treatment of CHD and may offer new target genes for prenatal screening of CHD.This article summarizes recent advances in the role of cilia in heart development and CHD pathogenesis and its mechanisms.

[Chemistry research on natural product derivatives of dihydro-ß-agarofuran sesquiterpenoids].

Dihydro-ß-agarofuran sesquiterpenoids possess chemical diversity and biodiversity. A dihydro-ß-agarofuran sesquiterpenoid with only hydroxyl groups has been prepared by basic hydrolysis of crude extract of Euonymus bungeanus with 0.4% yield. Twelve derivatives were available in esterification, oxidation, decarboxylation, etc. Extensive ~1H-NMR,~(13)C-NMR, MS spectroscopic analyses and single-crystal X-ray diffraction analysis with Cu Kα radiation indicated that eleven derivatives were new compounds. The results will provide reference for chemistry study on natural product derivatives of dihydro-ß-agarofuran sesquiterpenoids.

The less weight loss due to modest food restriction drove more fat accumulation in striped hamsters refed with high-fat diet.

Food restriction (FR) has been commonly used to decrease body fat, reducing the risk of overweight in humans and animals. However, the lost weight has been shown to be followed by overweight when food restriction ends. It remains uncertain whether the weight loss drives the overweight, or not. In the present study, striped hamsters were restricted by 15%, 30% and 40% of ad libitum food intake for 2 weeks, followed by high-fat refeeding for 6 weeks (FR15%-Re, FR30%-Re and FR40%-Re). The hamsters in FR15%, FR30% and FR40% groups decreased by 21.1%, 37.8% and 50.0% in fat mass (P < 0.01), and 16.8%, 42.8% and 53.4% in leptin levels (P < 0.01) compared with the hamsters fed ad libitum. The FR15%-Re, FR30%-Re and FR40%-Re groups showed 77.0%, 37.2% and 23.7% more body fat than ad libitum group (P < 0.01). The FR15%-Re group showed considerable decreases in gene expression of arcuate nucleus co-expressing proopiomelanocortin (POMC), cocaine - and amphetamineregulated transcript (CART) and the long isoform of leptin receptor (LepRb) in the hypothalamus and of several genes associated with fatty acid transport to mitochondria and ß-oxidation in brown adipose tissue and liver. It suggests that less weight loss is likely to drive more fat accumulation when food restriction ends, in which the impaired function of LepRb, POMC and CART in the brain and fatty acid oxidation in brown adipose tissue and liver may be involved.

Halorussus salinus sp. nov., isolated from a marine solar saltern.

A halophilic archaeal strain YJ-37-HT was isolated from Yangjiang marine solar saltern, China. Cells were pleomorphic rods, stained Gram negative and formed red-pigmented colonies on agar plate. Strain YJ-37-HT was able to grow at 20-50 °C (optimum 37 °C), at 0.9-4.8 M NaCl (optimum 2.6 M NaCl), at 0-1.0 M MgCl2 (optimum 0.3 MgCl2) and at pH 6.5-9.0 (optimum pH 7.0). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was found to be 5 % (w/v). The 16S rRNA gene and rpoB' gene of strain YJ-37-HT were phylogenetically related to the corresponding genes of Halorussus members (93.2-95.8 % and 90.1-93.9 % similarities, respectively). The major polar lipids of the strain were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and five glycolipids, sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), galactosyl mannosyl glucosyl diether (TGD-1), sulfated mannosyl glucosyl diether (S-DGD-1), mannosyl glucosyl diether (DGD-1) and diglycosyl diether (DGD-2). The DNA G+C content of strain YJ-37-HT was 64.9 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain YJ-37-HT (=CGMCC 1.12571T = JCM 30032T) represents a new species of Halorussus, for which the name Halorussus salinus sp. nov. is proposed.

Salinigranum salinum sp. nov., isolated from a marine solar saltern.

An extremely halophilic archaeal strain YJ-50-S2T was isolated from Yangjiang marine solar saltern, China. Cells were pleomorphic, stained Gram-negative and formed red-pigmented colonies on agar plates. Strain YJ-50-S2T was able to grow at 25-50 °C (optimum 37 °C), with 0.9-4.8 M NaCl (optimum 2.6 M NaCl) and 0-1.0 M MgCl2 (optimum 0.03 M MgCl2), and at pH 5.0-9.5 (optimum pH 7.5). The cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 5 % (w/v). The 16S rRNA gene and rpoB' gene of strain YJ-50-S2T were phylogenetically related to the corresponding genes of Salinigranum rubrum GX10T (97.0 % and 90.5 % similarities, respectively). The major polar lipids of strainYJ-50-S2T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and two major glycolipids chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. Several unidentified glycolipids were also detected. The DNA G+C content of strain YJ-50-S2T was 65.2 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain YJ-50-S2Trepresents a novel species of the genus Salinigranum, for which the name Salinigranum salinum sp. nov. is proposed. The type strain is YJ-50-S2T (=CGMCC 1.12572T=JCM 30033T).

Halomarina salina sp. nov., isolated from a marine solar saltern.

A halophilic archaeal strain, designated ZS-57-S(T), was isolated from Zhoushan marine solar saltern, China. Cells were observed to be pleomorphic, stained Gram-negative and formed red pigmented colonies on agar plates. Optimal growth was obtained at 3.9 M NaCl (range 1.4-4.8 M), 0.3 M MgCl2 (range 0-1.0 M), 30 °C (range 20-55 °C) and pH 6.5-7.5 (range 5.5-9.0). The cells were found to lyse in distilled water and the minimal NaCl concentration to prevent cell lysis was determined to be 5 % (w/v). The major polar lipids were identified as C20C20 and C20C25 diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, glucosyl mannosyl glucosyl diether and two unidentified glycolipids. The 16S rRNA gene and rpoB' gene of strain ZS-57-S(T) were phylogenetically related to the corresponding genes of Halomarina oriensis JCM 16495(T) (98.2 and 93.7 % similarities, respectively). The DNA G+C content of strain ZS-57-S(T) was determined to be 67.1 mol% (T m). The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain ZS-57-S(T) (=CGMCC 1.12543(T) = JCM 30039(T)) represents a new species of the genus Halomarina, for which the name Halomarina salina sp. nov. is proposed.

Halorubrum rutilum sp. nov. isolated from a marine solar saltern.

A halophilic archaeal strain, YJ-18-S1(T), was isolated from Yangjiang marine solar saltern, Guangxi Province, China. Cells were pleomorphic, stained Gram-negative and formed red-pigmented colonies on agar plates. Strain YJ-18-S1(T) was able to grow at 20-55 °C (optimum 37 °C), at 0.9-4.8 M NaCl (optimum 2.6 M NaCl), at 0.005-1.0 M MgCl2 (optimum 0.3 MgCl2) and at pH 5.5-8.5 (optimum pH 7.0). The cells were lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was found to be 5 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The 16S rRNA gene and rpoB' gene of strain YJ-18-S1(T) were phylogenetically related to the corresponding genes of Halorubrum members (94.3-98.0 and 86.7-96.1 % similarities, respectively). The DNA G+C content of strain YJ-18-S1(T) was 66.2 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain YJ-18-S1(T) (=CGMCC 1.12554(T) = JCM 30030(T)) represents a new species of Halorubrum, for which the name Halorubrum rutilum sp. nov. is proposed.