Molecular Pathophysiology of Congenital Long QT Syndrome.

Ion channels represent the molecular entities that give rise to the cardiac action potential, the fundamental cellular electrical event in the heart. The concerted function of these channels leads to normal cyclical excitation and resultant contraction of cardiac muscle. Research into cardiac ion channel regulation and mutations that underlie disease pathogenesis has greatly enhanced our knowledge of the causes and clinical management of cardiac arrhythmia. Here we review the molecular determinants, pathogenesis, and pharmacology of congenital Long QT Syndrome. We examine mechanisms of dysfunction associated with three critical cardiac currents that comprise the majority of congenital Long QT Syndrome cases: 1) IKs, the slow delayed rectifier current; 2) IKr, the rapid delayed rectifier current; and 3) INa, the voltage-dependent sodium current. Less common subtypes of congenital Long QT Syndrome affect other cardiac ionic currents that contribute to the dynamic nature of cardiac electrophysiology. Through the study of mutations that cause congenital Long QT Syndrome, the scientific community has advanced understanding of ion channel structure-function relationships, physiology, and pharmacological response to clinically employed and experimental pharmacological agents. Our understanding of congenital Long QT Syndrome continues to evolve rapidly and with great benefits: genotype-driven clinical management of the disease has improved patient care as precision medicine becomes even more a reality.

Drift calibration method of Fabry-Perot filters using two-stage decomposition and hybrid modeling.

Although tunable Fabry-Perot (F-P) filters are widely acknowledged as fiber Bragg grating (FBG) demodulators, F-P filters exhibit drift error when subjected to ambient temperature and piezo-electrical transducer (PZT) hysteresis. To address the drift issue, the majority of the existing literature makes use of additional devices like the F-P etalon and gas chamber. In this study, a novel drift calibration method based on two-stage decomposition and hybrid modeling is proposed. The initial drift error sequences are broken down into three frequency components using the variational mode decomposition (VMD), and the medium-frequency components are further broken down using the secondary VMD. The initial drift error sequences are significantly simplified by the two-stage VMD. On this foundation, the long short-term memory (LSTM) network and polynomial fitting (PF) are used to forecast the low-frequency and high-frequency drift errors, respectively. The LSTM enables the prediction of intricate nonlinear local behaviors, while the PF method predicts the overall trend. The benefits of LSTM and PF can be effectively utilized in this manner. Compared to the single-stage decomposition, two-stage decomposition achieves superior results. The suggested method is an affordable and effective alternative to the current drift calibration techniques.

Keratinocytes: innate immune cells in atopic dermatitis.

The skin is a unique immune organ that constitutes a complex network of physical, chemical and microbiological barriers against external insults. Keratinocytes are the most abundant cell type in the epidermis. These cells form the physical skin barrier and represent the first line of the host defense system by sensing pathogens via innate immune receptors, initiating anti-microbial responses and producing various cytokines, chemokines and anti-microbial peptides, which are important events in immunity. A damaged epidermal barrier in atopic dermatitis allows the penetration of potential allergens and pathogens to activate keratinocytes. Among the dysregulation of immune responses in atopic dermatitis, activated keratinocytes play a role in several biological processes that contribute to the pathogenesis of atopic dermatitis. In this review, we summarize the current understanding of the innate immune functions of keratinocytes in the pathogenesis of atopic dermatitis, with a special emphasis on skin-derived anti-microbial peptides and atopic dermatitis-related cytokines and chemokines in keratinocytes. An improved understanding of the innate immunity mediated by keratinocytes can provide helpful insight into the pathophysiological processes of atopic dermatitis and support new therapeutic efforts.

Hysteresis and temperature drift compensation for FBG demodulation by utilizing adaptive weight least square support vector regression.

Hysteresis and temperature drift deteriorate the demodulation performance of tunable Fabry-Perot (F-P) filters. This study addresses a novel adaptive weight least square support vector regression (AWLSSVR) to compensate for the hysteresis and temperature drift of F-P filters. The temperature drift of a referent fiber Bragg grating(FBG) is used to estimate the temperature drifts of other three sensing FBGs, and a novel adaptive weighting strategy with an asymmetric noise interval is proposed, to eliminate the effects of noise in the training dataset. The experimental results show that when the temperature-changing modes of the training and testing datasets were close to each other, the error of the proposed method is reduced to 8.7 pm, while the errors of the other three conventional methods based on LSSVR are more than 10.8 pm. Further, when the temperature-changing modes of the training and testing datasets were partly different, the error of the proposed method was reduced to 5.4 pm, while the errors of other methods were more than 11.9 pm. It was verified experimentally that the proposed AWLSSVR method is more accurate and robust than other versions of WLSSVR for training samples with noise, requires no additional hardware, and covers the entire C band.

Chemoenzymatic Total Synthesis of GM3 Gangliosides Containing Different Sialic Acid Forms and Various Fatty Acyl Chains.

Gangliosides are sialic acid-containing glycosphingolipids that have been found in the cell membranes of all vertebrates. Their important biological functions are contributed by both the glycan and the ceramide lipid components. GM3 is a major ganglioside and a precursor for many other more complex gangliosides. To obtain structurally diverse GM3 gangliosides containing various sialic acid forms and different fatty acyl chains in low cost, an improved process was developed to chemically synthesize lactosyl sphingosine from an inexpensive l-serine derivative. It was then used to obtain GM3 sphingosines from diverse modified sialic acid precursors by an efficient one-pot multienzyme sialylation system containing Pasteurella multocida sialyltransferase 3 (PmST3) with in situ generation of sugar nucleotides. A highly effective chemical acylation and facile C18-cartridge purification process was then used to install fatty acyl chains of varying lengths and different modifications. The chemoenzymatic method represents a powerful total synthetic strategy to access a library of structurally defined GM3 gangliosides to explore their functions.

Microarray analyses of closely related glycoforms reveal different accessibilities of glycan determinants on N-glycan branches.

Glycans mediate a wide variety of biological roles via recognition by glycan-binding proteins (GBPs). Comprehensive knowledge of such interaction is thus fundamental to glycobiology. While the primary binding feature of GBPs can be easily uncovered by using a simple glycan microarray harboring limited numbers of glycan motifs, their fine specificities are harder to interpret. In this study, we prepared 98 closely related N-glycoforms that contain 5 common glycan epitopes which allowed the determination of the fine binding specificities of several plant lectins and anti-glycan antibodies. These N-glycoforms differ from each other at the monosaccharide level and were presented in an identical format to ensure comparability. With the analysis platform we used, it was found that most tested GBPs have preferences toward only one branch of the complex N-glycans, and their binding toward the epitope-presenting branch can be significantly affected by structures on the other branch. Fine specificities described here are valuable for a comprehensive understanding and applications of GBPs.

Amplification and Preparation of Cellular O-Glycomes for Functional Glycomics.

Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N3-Bn-GalNAc(Ac)3 as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory. Cultured human non-small cell lung cancer (NSCLC) A549 cells take up the precursor, which is extended by cellular glycosyltransferases to produce 4-N3-Bn-α-O-glycans that are secreted into the culture medium. The O-glycan derivatives can be clicked with a fluorescent bifunctional tag that allows multidimensional HPLC purification and production of a tagged glycan library, representing the O-glycome of the corresponding cells. We obtained ∼5% conversion of precursor to O-glycans and purified a tagged O-glycan library of over 100 O-glycan derivatives, many of which were present in >100 nmol amounts and were sequenced by sequential MS fragmentation (MSn). These O-glycans were successfully printed onto epoxy glass slides as an O-glycome shotgun microarray. We used this novel array to explore binding activity of serum IgM in healthy persons and NSCLC patients at different cancer stages. This novel strategy provides access to complex O-glycans in significant quantities and may offer a new route to discovery of potential diagnostic disease biomarkers.

Diagnostic potential of urinary monocyte chemoattractant protein-1 for Alzheimer's disease and amnestic mild cognitive impairment.

BACKGROUND AND PURPOSE: The chemokine monocyte chemoattractant protein-1 (MCP-1) is involved in the pathogenesis of Alzheimer's disease (AD). This study aimed to investigate whether urinary MCP-1 can distinguish patients with AD, patients with amnestic mild cognitive impairment (aMCI) and cognitively normal (CN) subjects. METHODS: A total of 754 participants, including 97 patients with AD, 50 patients with aMCI and 84 age- and sex-matched CN controls as well as a cohort of 523 CN subjects of different ages, were enrolled from five hospitals located in different areas of China. Urinary MCP-1 levels were determined using enzyme-linked immunosorbent assays. The correlations between urinary MCP-1 levels and cognition test scores or age were analysed. The optimal diagnostic sensitivity and specificity were determined using receiver operating characteristic curve analysis. RESULTS: In the cohort of CN subjects of different ages, urinary MCP-1 levels increased with ageing and were correlated with age. The urinary MCP-1 levels were higher in females than in males. In the cohort composed of patients with AD, aMCI and age- and sex-matched CN controls, urinary MCP-1 levels were significantly higher in patients with AD and aMCI than in CN controls. There were no differences in urine MCP-1 levels between the AD group and the aMCI group. The urinary MCP-1 levels were correlated with the Mini-Mental State Examination scores and age, and were able to differentiate patients with AD and aMCI from CN subjects. CONCLUSIONS: Urinary MCP-1 is a potential biomarker for the diagnosis of AD and aMCI.

MicroRNA-506-3p inhibits proliferation and promotes apoptosis in ovarian cancer cell via targeting SIRT1/AKT/FOXO3a signaling pathway.

Ovarian cancer (OC) is one of the most common tumors in females. Growing evidence shows that microRNA-506-3p (miR-506-3p) is downregulated in OC tissues. The purpose of this study was to investigate the mechanism of miR-506-3p in modulating OC. Quantitative reverse transcriptase PCR (qRT-PCR) was employed to investigate the expression of miR-506-3p and its target in OC tissues or cell lines. CCK-8 or colony formation assay was used to examine cell viability or proliferation, respectively. Flow cytometry was demonstrated to detect cell apoptosis. Western blot was then applied to analyze underlying mechanisms. The potential target of miR-506-3p was examined via luciferase reporter assay. MiR-506-3p was significantly downregulated in both human OC tissues and cell lines. Overexpression of miR-506-3p not only decreased cell viability of OC cell lines but also promoted cell apoptosis, thus inhibiting OC progression. Moreover, SIRT1 (Sirtuin 1) was found to be a direct target of miR-506-3p, and SIRT1 expression was negatively regulated by miR-506-3p in OC cell lines. Further investigation revealed that overexpression of SIRT1 could promote cell viability as well as inhibit cell apoptosis, showing the reversed effect on OC progression compared to miR-506-3p. Lastly, AKT (Protein kinase B) /FOXO3a (Forkhead box O3) signaling pathway was inactivated by miR-506-3p while activated by SIRT1, relating to regulation of miR-506-3p on OC progression. Our results revealed a novel mechanism by which miR-506-3p inhibited proliferation while promoted apoptosis of OC via inactivation of SIRT1/AKT/FOXO3a signaling pathway, suggesting that miR-506-3p might be a potential target for OC.

Development and validation of new diagnostic criteria for atopic dermatitis in children of China.

BACKGROUND: In previous epidemiological study, the prevalence of atopic dermatitis (AD) was 12.94% among children aged 1-7 years by clinical diagnosis, whereas that was 4.76% and 3.51% using U.K., and Hanifin and Rajka diagnostic criteria. OBJECTIVE: We aimed to propose new diagnostic criteria for children and evaluate its efficiency in different populations. METHODS: We screened atopic features and analysed their correlation with AD using data from a previous study. A new set of diagnostic criteria for children in China was proposed and validated in 1031 children in outpatient clinics and 538 children in a birth cohort survey. Clinical diagnosis and atopic feature evaluation were performed face to face by dermatologists specialized in AD. Three criteria were compared for diagnostic efficiency using the clinical diagnosis as the reference. RESULTS: The new diagnostic criteria for children were based on (i) pruritus; (ii) 'typical morphology and distribution' or 'atypical morphology and distribution with xerosis'; and (iii) a chronic or chronically relapsing course. Compared to classical diagnostic criteria, the sensitivity of the new diagnostic criteria was significantly higher in the epidemiological survey and the clinical setting, especially obvious among mild and moderate AD. In the birth cohort, the new criteria showed similar sensitivity and specificity. CONCLUSION: The new criteria for children yielded higher sensitivity for the diagnosis of AD in the epidemiological survey and clinical setting, particularly for mild and moderate AD. Among the birth cohort with a complete medical history, three criteria showed similar sensitivity and specificity.

Machine-Driven Chemoenzymatic Synthesis of Glycopeptide.

Historically, researchers have put considerable effort into developing automation systems to prepare natural biopolymers such as peptides and oligonucleotides. The availability of such mature systems has significantly advanced the development of natural science. Over the past twenty years, breakthroughs in automated synthesis of oligosaccharides have also been achieved. A machine-driven platform for glycopeptide synthesis by a reconstructed peptide synthesizer is described. The designed platform is based on the use of an amine-functionalized silica resin to facilitate the chemical synthesis of peptides in organic solvent as well as the enzymatic synthesis of glycan epitopes in the aqueous phase in a single reaction vessel. Both syntheses were performed by a peptide synthesizer in a semiautomated manner.

Genetically Encoding Photocaged Quinone Methide to Multitarget Protein Residues Covalently in Vivo.

Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2 R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in Escherichia coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His, and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn, and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-cross-linking Uaas, and FnbY exhibited cross-linking efficiency higher than p-azido-phenylalanine. The photoactivatable and multitargeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.

Genetically Introducing Biochemically Reactive Amino Acids Dehydroalanine and Dehydrobutyrine in Proteins.

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-l-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intramolecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

Gamma radiation and thermal-induced effects on the spectral performance of BACs in Bi/Er codoped aluminosilicate fibers.

The results of γ-radiation (2-72 kGy) and thermal-induced effects on BACs in Bi/Er codoped aluminosilicate fibers (BEDF) have been presented first in this paper. We observed that the radiation effect on on-off gain and optical absorption associated with BAC-Al and BAC-Si was insignificant, while the effect on luminescence was considerable. However, the effect on luminescence is caused by the radiation-induced darkening, which is likely linked to thermal bleachable Al-OHC point defects generated by γ-radiation. We carried out the thermal experiment and observed thermal bleaching of the γ-irradiated fiber at a low temperature of 300 °C. The observations indicate that, while γ-radiation could introduce significant background loss, BAC-Al and BAC-Si are fairly radiation resistant. This is the first time that BACs show good radiation resistance in irradiated BEDFs.

[Red blood cell lifespan detected by endogenous carbon monoxide breath test in patients with polycythemia vera].

Objective: To detect the red blood cell lifespan in patients with polycythemia vera (PV), and explore the influencing factors. Methods: From February 2017 to December 2018, 27 patients with PV at Blood Diseases Hospital, Chinese Academy of Medical Science and 18 normal controls were recruited. Red blood cell lifespan was detected by endogenous carbon monoxide (CO) breath test. The related factors were analyzed. Results: The average red blood cell lifespan of 27 PV patients was 80 (range, 35-120) days (d), which was significantly shorter than that of the normal controls [110.5(69-166) d, P<0.05], namely 35.3 d shorter. The red blood cell lifespan of ten newly diagnosed patients and 17 patients who were treated with hydroxyurea and/or interferon were 98 (35-117) d and 69 (45-120) d, respectively, which were both shorter than that of the normal control (P=0.010, 0.000). Correlation analysis showed that red blood cell lifespan of patients with newly diagnosed PV was associated with JAK2 mutation allele burden (r=0.900, P=0.037), peripheral blood lymphocyte count (r=-0.742, P=0.014) and the level of serum vitamin B(12) (r=-0.821, P=0.023). Conclusion: The lifespan of red blood cells in patients with PV is about one-third shorter than normal, and is related to JAK2 mutation allele burden, absolute lymphocyte count, and serum vitamin B(12) level.

Production of homogeneous glycoprotein with multisite modifications by an engineered N-glycosyltransferase mutant.

Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N-glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity toward the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) N-glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.

Genetically Encoding Fluorosulfate-l-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo.

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics.

Streamlined chemoenzymatic total synthesis of prioritized ganglioside cancer antigens.

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacßSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.

[The clinical and laboratory characteristics of congenital pyruvate kinase deficiency].

Clinical data of 19 patients with congenital pyruvate kinase deficiency were analyzed. Insufficient pyruvate kinase confirmed the diagnosis. Laboratory parameters of hemolysis were summarized. In cases of neonatal hyperbilirubinemia and unexplained hemolytic anemia, pyruvate kinase activity and next generation sequencing test may help the early diagnosis.

[The mechanism of nicotine on human bronchial smooth muscle cell contraction].

Objective: To investigate the molecular mechanism of contractility dysfunction of human bronchial smooth muscle cells induced by nicotine. Methods: Primary human bronchial smooth muscle cells were cultured in vitro. The cells were divided into a control group and a nicotine group which was treated with 10(-5) mol/L nicotine for 48 h and transfected with or without α7nAChR-siRNA (The siNC group, siNC + nicotine group and siα7nAChR + nicotine group). The effects of nicotine on the cell contractile function were examined by collagen gel shrinkage assay. The expressions of α7nAChR and TRPC6 protein in nicotine-treated human bronchial smooth muscle cells were detected by Western blotting. The change of intracellular calcium concentration by nicotine was detected by calcium ion imaging system.Data were analyzed by t test or single factor analysis of variance. Results: The area of collagen gel in the nicotine group (24±8)% was significantly lower than that in the control group (59±14)% (t=3.78, P<0.05). Compared with the control group, the expression of α7nAChR protein in nicotine-induced group (173±16)% was significantly higher than that of controls 100±0)%, t=-6.848, P<0.05. Compared with the siNC group [(72±10)%, (0.79±0.07), (0.41±0.04) and (0.17±0.02) respectively], the collagen gel area of siNC + nicotine group was significantly reduced by (37±10)%. However, the basal calcium level (1.04±0.02), store operated calcium entry level (SOCE, 0.68±0.03) and receptor operated calcium entry level (ROCE, 0.36±0.02) were remarkably elevated in the nicotine treated group (all P<0.05). Furthermore, compared with siNC + nicotine group, the area of collagen gel in siα7nAChR + nicotine group was significantly increased (62±10)%, and the basal calcium level (0.78±0.06), SOCE level (0.39±0.05) and ROCE level (0.15±0.02) were significantly reduced (all P<0.05). Conclusions: Nicotine can increase the expression of TRPC6 protein, SOCE and ROCE level, and increase the intracellular calcium concentration by upregulating the expression of α7nAChR protein, thereby promoting smooth muscle cell contraction.