Functional Characterization of F3H Gene and Optimization of Dihydrokaempferol Biosynthesis in Saccharomyces cerevisiae.

The 1092 bp F3H gene from Trapa bispinosa Roxb., which was named TbF3H, was cloned and it encodes 363 amino acids. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbF3H with flavanone 3-hydroxylase from other plants. A functional analysis showed that TbF3H of Trapa bispinosa Roxb. encoded a functional flavanone 3-hydroxylase; it catalyzed the formation of dihydrokaempferol (DHK) from naringenin in S. cerevisiae. The promoter strengths were compared by fluorescence microscopy and flow cytometry detection of the fluorescence intensity of the reporter genes initiated by each constitutive promoter (FITC), and DHK production reached 216.7 mg/L by the promoter adjustment strategy and the optimization of fermentation conditions. The results presented in this study will contribute to elucidating DHK biosynthesis in Trapa bispinosa Roxb.

Metal transporter SLC39A14/ZIP14 modulates regulation between the gut microbiome and host metabolism.

Metal transporter SLC39A14/ZIP14 is localized on the basolateral side of the intestine, functioning to transport metals from blood to intestine epithelial cells. Deletion of Slc39a14/Zip14 causes spontaneous intestinal permeability with low-grade chronic inflammation, mild hyperinsulinemia, and greater body fat with insulin resistance in adipose. Importantly, antibiotic treatment reverses the adipocyte phenotype of Slc39a14/Zip14 knockout (KO), suggesting a potential gut microbial role in the metabolic alterations in the Slc39a14/Zip14 KO mice. Here, we investigated the hypothesis that increased intestinal permeability and subsequent metabolic alterations in the absence of Zip14 could be in part due to alterations in gut microbial composition. Dietary metals have been shown to be involved in the regulation of gut microbial diversity and composition. However, studies linking the action of intestinal metal transporters to gut microbial regulation are lacking. We showed the influence of deletion of metal transporter Slc39a14/Zip14 on gut microbiome composition and how ZIP14-linked changes to gut microbiome community composition are correlated with changes in host metabolism. Deletion of Slc39a14/Zip14 generated Zn-deficient epithelial cells and luminal content in the entire intestinal tract, a shift in gut microbial composition that partially overlapped with changes previously associated with obesity and inflammatory bowel disease (IBD), increased the fungi/bacteria ratio in the gut microbiome, altered the host metabolome, and shifted host energy metabolism toward glucose utilization. Collectively, our data suggest a potential predisease microbial susceptibility state dependent on host gene Slc39a14/Zip14 that contributes to intestinal permeability, a common trait of IBD, and metabolic disorders such as obesity and type 2 diabetes.NEW & NOTEWORTHY Metal dyshomeostasis, intestinal permeability, and gut dysbiosis are emerging signatures of chronic disorders, including inflammatory bowel diseases, type-2 diabetes, and obesity. Studies in reciprocal regulations between host intestinal metal transporters genes and gut microbiome are scarce. Our research revealed a potential predisease microbial susceptibility state dependent on the host metal transporter gene, Slc39a14/Zip14, that contributes to intestinal permeability providing new insight into understanding host metal transporter gene-microbiome interactions in developing chronic disease.

GGC Repeat Expansion of RILPL1 is Associated with Oculopharyngodistal Myopathy.

OBJECTIVE: Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ptosis, dysarthria, ophthalmoplegia, and distal muscle weakness. Recent studies revealed that GGC repeat expansions in 5'-UTR of LRP12, GIPC1, and NOTCH2NLC are associated with OPDM. Despite these advances, approximately 30% of OPDM patients remain genetically undiagnosed. Herein, we aim to investigate the genetic basis for undiagnosed OPDM patients in two unrelated Chinese Han families. METHODS: Parametric linkage analysis was performed. Long-read sequencing followed by repeat-primed polymerase chain reaction and amplicon length polymerase chain reaction were used to determine the genetic cause. Targeted methylation sequencing was implemented to detect epigenetic changes. The possible pathogenesis mechanism was investigated by quantitative polymerase chain reaction, immunoblotting, RNA fluorescence in situ hybridization, and immunofluorescence staining of muscle biopsy samples. RESULTS: The disease locus was mapped to 12q24.3. Subsequently, GGC repeat expansion in the promoter region of RILPL1 was identified in six OPDM patients from two families, findings consistent with a founder effect, designated as OPDM type 4. Targeted methylation sequencing revealed hypermethylation at the RILPL1 locus in unaffected individuals with ultralong expansion. Analysis of muscle samples showed no significant differences in RILPL1 mRNA or RILPL1 protein levels between patients and controls. Public CAGE-seq data indicated that alternative transcription start sites exist upstream of the RefSeq-annotated RILPL1 transcription start site. Strand-specific RNA-seq data revealed bidirectional transcription from the RILPL1 locus. Finally, fluorescence in situ hybridization/immunofluorescence staining showed that both sense and antisense transcripts formed RNA foci, and were co-localized with hnRNPA2B1 and p62 in the intranuclear inclusions of OPDM type 4 patients. INTERPRETATION: Our findings implicate abnormal GGC repeat expansions in the promoter region of RILPL1 as a novel genetic cause for OPDM, and suggest a methylation mechanism and a potential RNA toxicity mechanism are involved in OPDM type 4 pathogenesis. ANN NEUROL 2022;92:512-526.

Identification and characterization of 3-ketosphinganine reductase activity encoded at the BT_0972 locus in Bacteroides thetaiotaomicron.

Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive. In this study, we analyzed the 3-ketosphinganine reductase activity of several candidate proteins in Bacteroides thetaiotaomicron chosen based on sequence similarity to the yeast 3-ketosphinganine reductase gene. We further developed a metabolomics-based 3-ketosphinganine reductase activity assay, which revealed that a gene at the locus BT_0972 encodes a protein capable of converting 3-ketosphinganine to sphinganine. Taken together, these results provide greater insight into pathways for bacterial sphingolipid synthesis that can aid in future efforts to understand how microbial sphingolipid synthesis modulates host-microbe interactions.

CRISPR/Cas9-based genome editing for the modification of multiple duplications that cause Duchenne muscular dystrophy.

With the development of basic research, some genetic-based methods have been found to treat Duchenne muscular dystrophy (DMD) with large deletion mutations and nonsense mutations. Appropriate therapeutic approaches for repairing multiple duplications are limited. We used the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system with patient-derived primary myoblasts to correct multiple duplications of the dystrophin gene. Muscle tissues from a patient carrying duplications of dystrophin were obtained, and tissue-derived primary cells were cultured. Myoblasts were purified with an immunomagnetic sorting system using CD56 microbeads. After transduction by lentivirus with a designed single guide RNA (sgRNA) targeting a duplicated region, myoblasts were allowed to differentiate for 7 days. Copy number variations in the exons of the patient's myotubes were quantified by real-time PCR before and after genetic editing. Western blot analysis was performed to detect the full-length dystrophin protein before and after genetic editing. The ten sequences predicted to be the most likely off-targets were determined by Sanger sequencing. The patient carried duplications of exon 18-25, dystrophin protein expression was completely abrogated. Real-time PCR showed that the copy number of exon 25 in the patient's myotubes was 2.015 ± 0.079 compared with that of the healthy controls. After editing, the copy number of exon 25 in the patient's modified myotubes was 1.308 ± 0.083 compared with that of the healthy controls (P < 0.001). Western blot analysis revealed no expression of the dystrophin protein in the patient's myotubes before editing. After editing, the patient's myotubes expressed the full-length dystrophin protein at a level that was ~6.12% of that in the healthy control samples. Off-target analysis revealed no abnormal editing at the ten sites predicted to be the most likely off-target sites. The excision of multiple duplications by the CRISPR/Cas9 system restored the expression of full-length dystrophin. This study provides proof of evidence for future genome-editing therapy in patients with DMD caused by multiple duplication mutations.

Human poliovirus receptor contributes to biliary atresia pathogenesis by exacerbating natural-killer-cell-mediated bile duct injury.

BACKGROUND AND AIMS: Natural killer (NK) cells play an important role in biliary atresia (BA) pathogenesis; human poliovirus receptor (PVR) is an important NK-cell modulator. Here, we explored the role of PVR in BA pathogenesis. METHODS: Poliovirus receptor expression and NK cell-associated genes were detected in human BA samples and a rotavirus-induced BA mouse model using quantitative PCR and immunofluorescence staining. Chemically modified small interfering RNA silenced PVR expression in the BA model, and its effects on the population and function of intrahepatic NK cells were investigated using flow cytometry (FCM). The effects of PVR overexpression and knockdown on proliferation, apoptosis and NK-cell-mediated lysis of cultured human cholangiocytes were analysed using FCM and cell viability assays. Serum PVR, high-mobility group box 1 (HMGB1), and interleukin-1beta (IL-1beta) levels were measured in a cohort of 50 patients using ELISA. RESULTS: Poliovirus receptor expression was upregulated in the biliary epithelium of BA patients and BA model and was positively correlated with the population and activation of intrahepatic NK cells. Silencing of PVR expression impaired the cytotoxicity of NK cells, reduced inflammation and protected mice from rotavirus-induced BA. Activation of the TLR3-IRF3 signalling pathway induced PVR expression in cultured cholangiocytes. PVR overexpression promoted proliferation and inhibited the apoptosis of cholangiocytes but exacerbated NK cell-mediated cholangiocyte lysis. Serum PVR levels were elevated in BA patients and were positively correlated with HMGB1 and IL-1beta levels. CONCLUSIONS: Poliovirus receptor contributes to BA pathogenesis by regulating NK cell-mediated bile duct injury; PVR has the value as a biomarker of BA.

TSAd Plays a Major Role in Myo9b-Mediated Suppression of Malignant Pleural Effusion by Regulating TH1/TH17 Cell Response.

Emerging evidence indicates that Myo9b is a cancer metastasis-related protein and functions in a variety of immune-related diseases. However, it is not clear whether and how Myo9b functions in malignant pleural effusion (MPE). In this study, our data showed that Myo9b expression levels correlated with lung cancer pleural metastasis, and nucleated cells in MPE from either patients or mice expressed a lower level of Myo9b than those in the corresponding blood. Myo9b deficiency in cancer cells suppressed MPE development via inhibition of migration. Myo9b deficiency in mice suppressed MPE development by decreasing TH1 cells and increasing TH17 cells. CD4+ naive T cells isolated from Myo9b-/- mouse spleens exhibited less TH1 cell differentiation and more TH17 cell differentiation in vitro. mRNA sequencing of nucleated cells showed that T cell-specific adaptor protein (TSAd) was downregulated in Myo9b-/- mouse MPE, and enrichment of the H3K27me3 mark in the TSAd promoter region was found in the Myo9b-/- group. Naive T cells purified from wild type mouse spleens transfected with TSAd-specific small interfering RNAs (siRNAs) also showed less TH1 cell differentiation and more TH17 cell differentiation than those from the siRNA control group. Furthermore, downregulation of TSAd in mice using cholesterol-conjugated TSAd-specific siRNA suppressed MPE development, decreased TH1 cells, and increased TH17 cells in MPE in vivo. Taken together, Myo9b deficiency suppresses MPE development not only by suppressing pleural cancer metastasis but also by regulating TH1/TH17 cell response via a TSAd-dependent pathway. This work suggests Myo9b and TSAd as novel candidates for future basic and clinical investigations of cancer.

Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from Trapa bispinosa Roxb.

Trapa bispinosa Roxb. is an economical crop for medicine and food. Its roots, stems, leaves, and pulp have medicinal applications, and its shell is rich in active ingredients and is considered to have a high medicinal value. One of the main functional components of the Trapa bispinosa Roxb. shell is 1-galloyl-beta-D-glucose (ßG), which can be used in medical treatment and is also an essential substrate for synthesizing the anticancer drug beta-penta-o-Galloyl-glucosen (PGG). Furthermore, gallate 1-beta-glucosyltransferase (EC 2.4.1.136) has been found to catalyze gallic acid (GA) and uridine diphosphate glucose (UDPG) to synthesize ßG. In our previous study, significant differences in ßG content were observed in different tissues of Trapa bispinosa Roxb. In this study, Trapa bispinosa Roxb. was used to clone 1500 bp of the UGGT gene, which was named TbUGGT, to encode 499 amino acids. According to the specificity of the endogenous expression of foreign genes in Escherichia coli, the adaptation codon of the cloned original genes was optimized for improved expression. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbUGGT with squalene synthases from other plants. The TbUGGT gene was constructed into a PET-28a expression vector and then transferred into Escherichia coli Transsetta (DE3) for expression. The recombinant protein had a molecular weight of 55 kDa and was detected using SDS-PAGE. The proteins were purified using multiple fermentation cultures to simulate the intracellular environment, and a substrate was added for in vitro reaction. After the enzymatic reaction, the levels of ßG in the product were analyzed using HPLC and LC-MS, indicating the catalytic activity of TbUGGT. The cloning and functional analysis of TbUGGT may lay the foundation for further study on the complete synthesis of ßG in E. coli.

Structure-Based Bioisosterism Design, Synthesis, Biological Activity and Toxicity of 1,2,4-Oxadiazole Substituted Benzamides Analogues Containing Pyrazole Rings.

In order to discover pesticidal lead compounds with high activity and low toxicity, a series of novel benzamides substituted with pyrazole-linked 1,2,4-oxadiazole were designed via bioisosterism. The chemical structures of the target compounds were confirmed via 1H NMR, 13C NMR and HRMS analysis. The preliminary bioassay showed that most compounds exhibited good lethal activities against Mythimna separate, Helicoverpa armigera, Ostrinia nubilalis and Spodoptera frugiperda at 500 mg/L. Particularly in the case of Mythimna separate, compound 14q (70%) exhibited obvious insecticidal activity. In addition, compound 14h demonstrated good fungicidal activity against Pyricularia oryae with an inhibition rate of 77.8%, and compounds 14e, 14k, 14n and 14r also showed certain antifungal activities (55.6-66.7%). The zebrafish toxicity test showed that the LC50 of compound 14h was 14.01 mg/L, which indicated that it may be used as a potential leading compound for further structural optimization.

Benzamides Substituted with Quinoline-Linked 1,2,4-Oxadiazole: Synthesis, Biological Activity and Toxicity to Zebrafish Embryo.

To develop new compounds with high activity, broad spectrum and low-toxicity, 17 benzamides substituted with quinoline-linked 1,2,4-oxadiazole were designed using the splicing principle of active substructures and were synthesized. The biological activities were evaluated against 10 fungi, indicating that some of the synthetic compounds showed excellent fungicidal activities. For example, at 50 mg/L, the inhibitory activity of 13p (3-Cl-4-Cl substituted, 86.1%) against Sclerotinia sclerotiorum was superior to that of quinoxyfen (77.8%), and the inhibitory activity of 13f (3-CF3 substituted, 77.8%) was comparable to that of quinoxyfen. The fungicidal activities of 13f and 13p to Sclerotinia sclerotiorum were better than that of quinoxyfen (14.19 mg/L), with EC50 of 6.67 mg/L and 5.17 mg/L, respectively. Furthermore, the acute toxicity of 13p was 19.42 mg/L, classifying it as a low-toxic compound.

Dietary sphinganine is selectively assimilated by members of the mammalian gut microbiome.

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet-microbiome interactions. Here, we used a click chemistry-based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine [sphinganine alkyne (SAA)] into the murine gut microbial community (bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet-microbiome interactions.

IL-10 promotes malignant pleural effusion by regulating TH 1 response via an miR-7116-5p/GPR55/ERK pathway in mice.

IL-10, produced by a wide variety of cells, is a highly pleiotropic cytokine that plays a critical role in the control of immune responses. However, its regulatory activity in tumor immunity remains poorly understood. In this study, we report that IL-10 deficiency robustly suppressed the formation of malignant pleural effusion (MPE) and significantly enhanced miR-7116-5p expression in pleural CD4+ T cells. We demonstrated that miR-7116-5p suppressed IL-10-mediated MPE formation by inhibiting pleural vascular permeability as well as tumor angiogenesis and tumor growth. IL-10 promoted MPE formation by suppressing miR-7116-5p that enhances TH 1 response. We identified G protein-coupled receptor 55 (GPR55) as a potential target of miR-7116-5p, and miR-7116-5p promoted TH 1 cell function by downregulating GPR55. Moreover, GPR55 promoted MPE formation by inhibiting TH 1 cell expansion through the ERK phosphorylation pathway. These results uncover an IL-10-mediated pathway controlling TH 1 cells and demonstrate a central role for miR-7116-5p/GPR55/ERK signaling in the physiological regulation of IL-10-driven pro-malignant responses.

CHIP control degradation of mutant ETF:QO through ubiquitylation in late-onset multiple acyl-CoA dehydrogenase deficiency.

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common form of lipid storage myopathy. The disease is mainly caused by mutations in electron-transfer flavoprotein dehydrogenase gene (ETFDH), which leads to decreased levels of ETF:QO in skeletal muscle. However, the specific underlying mechanisms triggering such degradation remain unknown. We constructed expression plasmids containing wild type ETF:QO and mutants ETF:QO-A84T, R175H, A215T, Y333C, and cultured patient-derived fibroblasts containing the following mutations in ETFDH: c.250G>A (p.A84T), c.998A>G (p.Y333C), c.770A>G (p.Y257C), c.1254_1257delAACT (p. L418TfsX10), c.524G>A (p.R175H), c.380T>A (p.L127P), and c.892C>T (p.P298S). We used in vitro expression systems and patient-derived fibroblasts to detect stability of ETF:QO mutants then evaluated their interaction with Hsp70 interacting protein CHIP with active/inactive ubiquitin E3 ligase carboxyl terminus using western blot and immunofluorescence staining. This interaction was confirmed in vitro and in vivo by co-immunoprecipitation and immunofluorescence staining. We confirmed the existence two ubiquitination sites in mutant ETF:QO using mass spectrometry (MS) analysis. We found that mutant ETF:QO proteins were unstable and easily degraded in patient fibroblasts and in vitro expression systems by ubiquitin-proteasome pathway, and identified the specific ubiquitin E3 ligase as CHIP, which forms complex to control mutant ETF:QO degradation through poly-ubiquitination. CHIP-dependent degradation of mutant ETF:QO proteins was confirmed by MS and site-directed mutagenesis of ubiquitination sites. Hsp70 is directly involved in this process as molecular chaperone of CHIP. CHIP plays an important role in ubiquitin-proteasome pathway dependent degradation of mutant ETF:QO by working as a chaperone-assisted E3 ligase, which reveals CHIP's potential role in pathological mechanisms of late-onset MADD.

Conformational Sensing by a Mammalian Olfactory Receptor.

To identify odors, the mammalian nose deploys hundreds of olfactory receptors (ORs) from the rhodopsin-like class of the G protein-coupled receptor superfamily. Odorants having multiple rotatable bonds present a problem for the stereochemical shape-based matching process assumed to govern the sense of smell through OR-odorant recognition. We conformationally restricted the carbon chain of the odorant octanal to ask whether an OR can respond differently to different odorant conformations. By using calcium imaging to monitor signal transduction in sensory neurons expressing the mouse aldehyde OR, Olfr2, we found that the spatial position of the C7 and C8 carbon atoms of octanal, in relation to its -CHO group, determines whether an aliphatic aldehyde functions as an agonist, partial agonist or antagonist. Our experiments provide evidence that an odorant can manipulate an OR through its intrinsic conformational repertoire, in unexpected analogy to the photon-controlled aldehyde manipulation observed in rhodopsin.

Missed nursing care in patient education: A qualitative study of different levels of nurses' perspectives.

AIM: To explore the different levels of nurses' perspectives in the delivery of patient education in postoperative care. BACKGROUND: Patient education is a frequently reported missed nursing care and can lead to postoperative complications and hospital readmissions. METHODS: Descriptive exploratory qualitative study involving eight focus groups with 35 nurses was conducted in an acute hospital. Interviews were audio-recorded and transcribed verbatim. Data were thematically analysed. RESULTS: The analysis yielded three themes: 'Role ambiguity' between the levels of nurses concerning their roles in patient education; 'Not a priority nursing care' for patient education due to competing work demands and the missing workplace culture to teach; and 'Informal teaching' carried out conversationally during nursing care activities. CONCLUSION: This study augments the need to develop strategies, including informal teaching, to strengthen the delivery of patient education to avert missed nursing care. IMPLICATIONS FOR NURSING MANAGEMENT: Nurse managers and educators are instrumental in establishing role clarity between ward nurses and specialty care nurses for patient education, recognizing patient education as the next nurse-sensitive indicator in reflecting quality of care, fostering positive workplace cultures to teach and providing ward nurses with trainings on communication strategies to provide effective informal teaching at bedside.

[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.

Transcription Profiles Reveal the Regulatory Synthesis of Phenols during the Development of Lotus Rhizome (Nelumbo nucifera Gaertn).

Lotus (Nelumbo nucifera Gaertn) is a wetland vegetable famous for its nutritional and medicinal value. Phenolic compounds are secondary metabolites that play important roles in the browning of fresh-cut fruits and vegetables, and chemical constituents are extracted from lotus for medicine due to their high antioxidant activity. Studies have explored in depth the changes in phenolic compounds during browning, while little is known about their synthesis during the formation of lotus rhizome. In this study, transcriptomic analyses of six samples were performed during lotus rhizome formation using a high-throughput tag sequencing technique. About 23 million high-quality reads were generated, and 92.14% of the data was mapped to the reference genome. The samples were divided into two stages, and we identified 23,475 genes in total, 689 of which were involved in the biosynthesis of secondary metabolites. A complex genetic crosstalk-regulated network involved in the biosynthesis of phenolic compounds was found during the development of lotus rhizome, and 25 genes in the phenylpropanoid biosynthesis pathway, 18 genes in the pentose phosphate pathway, and 30 genes in the flavonoid biosynthesis pathway were highly expressed. The expression patterns of key enzymes assigned to the synthesis of phenolic compounds were analyzed. Moreover, several differentially expressed genes required for phenolic compound biosynthesis detected by comparative transcriptomic analysis were verified through qRT-PCR. This work lays a foundation for future studies on the molecular mechanisms of phenolic compound biosynthesis during rhizome formation.

Effects of a Lysine-Involved Maillard Reaction on the Structure and In Vitro Activities of Polysaccharides from Longan Pulp.

The effects of amino acid-involved Maillard reactions (MRs) on the structure and activities of longan pulp polysaccharides (LPs), which were heteropolysaccharides mainly composed of glucose, galactose, mannose, rhamnose, glucuronic acid, ribose, and galacturonic acid, were investigated. The changes of browning degree and molecular weight (Mw) distribution in the MR systems containing LPs and amino acids (lysine, proline, or glycine) indicated that lysine was more active in conjugating with LPs. The MR-modified LPs (MLPs) obtained via a 4 h MR between LPs and lysine showed obvious structural differences from LPs. Specifically, particle-like LPs contained 94% fractions with a Mw less than 7.07 kDa, by contrast, network-like MLPs contained 45% fractions with a Mw larger than 264.1 kDa. Moreover, MLPs showed stronger radical scavenging abilities and macrophage immunostimulating effects, but weaker cancer cell growth-inhibitory abilities. The results indicate that the amino acid-involved MR is a promising method to modify native polysaccharides for better biological properties.

Role transition: A descriptive exploratory study of assistant nurse clinicians in Singapore.

AIM: To explore the role-transition experiences of assistant nurse clinicians after their first year of appointment. BACKGROUND: The National Nursing Taskforce was set up in Singapore to examine the professional development and recognition of nurses. It created the assistant nurse clinician role as an avenue for the nurses' career development. The role was intended to assist nurse managers to guide the nursing team in the assessment, planning, and delivery of patient care. METHODS: A qualitative descriptive study design was adopted. A purposive sample of 22 registered nurses from six acute care institutions and two polyclinics in Singapore participated in the face-to-face interviews. An inductive content analysis approach was used to analyse the data. RESULTS: Four themes emerged: (a) promotion to assistant nurse clinician is a form of recognition and vindication; (b) there was uncertainty about the expected role of the assistant nurse clinician; (c) experience eases transition; and (d) there was a need for peer support, mentorship, and training. CONCLUSIONS: The job description of the assistant nurse clinician needs to be better defined to provide greater clarity about their clinical and administrative duties and what is expected of their performance. IMPLICATIONS FOR NURSING MANAGEMENT: It is essential for nurse managers to provide successful role-transition strategies to help the newly appointed assistant nurse clinicians to become efficient and effective leaders.