- Isolation, purification, characterization and direct conjugation of the lipid A-free lipopolysaccharide of Vibrio cholerae O139. [Journal Article]
- CChemistry 2019 Jul 15
- The lipopolysaccharide (LPS) of Vibrio cholerae O139, strain CIRS245, was isolated conventionally, and the lipid A was removed by mild acid hydrolysis (0.1 M NaOAc buffer containing 1% SDS, pH 4.2, 9…
The lipopolysaccharide (LPS) of Vibrio cholerae O139, strain CIRS245, was isolated conventionally, and the lipid A was removed by mild acid hydrolysis (0.1 M NaOAc buffer containing 1% SDS, pH 4.2, 95°C, 8 h). The crude product was a complex mixture consisting mainly of constituent fragments of the O-specific polysaccharide-core (OSPc). The OSPc was only a minor component in the mixture. Two-stage purification of the crude OSPc by HPLC gave pure OSPc fragment of the LPS, as shown by NMR spectroscopy, analytical HPLC and ESI-MS. This material is the purest OSPc fragment of the LPS from Vibrio cholerae O139 reported to date. The purified OSPc was readily converted to the corresponding methyl squarate derivative and the latter was conjugated to BSA. The conjugate, when examined by ELISA, showed immunoreactivity with sera from patients in Bangladesh recovering from cholera caused by V. cholerae O139, but not O1.
- Replisome structure suggests mechanism for continuous fork progression and post-replication repair. [Review]
- DRDNA Repair (Amst) 2019 Jul 08; :102658
- What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA polymerase, and thus the replicati…
What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA polymerase, and thus the replication-fork progression, would stall at road blocks. After the discovery of replication-fork helicase and replication re-initiation factors by the 1990s, it became clear that the replisome can "skip" impasses and finish replication with single-stranded gaps and double-strand breaks in the product DNA. But the mechanism for continuous fork progression after encountering roadblocks is entangled with translesion synthesis, replication fork reversal and recombination repair. The recently determined structure of the bacteriophage T7 replisome offers the first glimpse of how helicase, primase, leading-and lagging-strand DNA polymerases are organized around a DNA replication fork. The tightly coupled leading-strand polymerase and lagging-strand helicase provides a scaffold to consolidate data accumulated over the past five decades and offers a fresh perspective on how the replisome may skip lesions and complete discontinuous DNA synthesis. Comparison of the independently evolved bacterial and eukaryotic replisomes suggests that repair of discontinuous DNA synthesis occurs post replication in both.
- A gene expression network analysis of the pancreatic islets from lean and obese mice identifies complement 1q like-3 secreted protein as a regulator of β-cell function. [Journal Article]
- SRSci Rep 2019 Jul 12; 9(1):10119
- Secreted proteins are important metabolic regulators. Identifying and characterizing the role of secreted proteins from small tissue depots such as islets of Langerhans, which are required for the pr…
Secreted proteins are important metabolic regulators. Identifying and characterizing the role of secreted proteins from small tissue depots such as islets of Langerhans, which are required for the proper control of whole-body energy metabolism, remains challenging. Our objective was to identify islet-derived secreted proteins that affect islet function in obesity. Lean and obese mouse islet expression data were analyzed by weighted gene co-expression network analysis (WGCNA) to identify trait-associated modules. Subsequently, genes within these modules were filtered for transcripts that encode for secreted proteins based on intramodular connectivity, module membership, and differential expression. Complement 1q like-3 (C1ql3) secreted protein was identified as a hub gene affecting islet function in obesity. Co-expression network, hierarchal clustering, and gene-ontology based approaches identified a putative role for C1ql3 in regulating β-cell insulin secretion. Biological validation shows that C1ql3 is expressed in β-cells, it inhibits insulin secretion and key genes that are involved in β-cell function. Moreover, the increased expression of C1ql3 is correlated with the reduced insulin secretion in islets of obese mice. Herein, we demonstrate a streamlined approach to effectively screen and determine the function of secreted proteins in islets, and identified C1ql3 as a putative contributor to reduced insulin secretion in obesity, linking C1ql3 to an increased susceptibility to type 2 diabetes.
- Acid Base Balance and Progression of Kidney Disease. [Review]
- SNSemin Nephrol 2019; 39(4):406-417
- A large body of work in animals and human beings supports the hypothesis that metabolic acidosis has a deleterious effect on the progression of kidney disease. Alkali therapy, whether pharmacological…
A large body of work in animals and human beings supports the hypothesis that metabolic acidosis has a deleterious effect on the progression of kidney disease. Alkali therapy, whether pharmacologically or through dietary intervention, appears to slow CKD progression, but an appropriately powered randomized controlled trial with a low risk of bias is required to reach a more definitive conclusion. Recent work on urinary ammonium excretion has shown that the development of prognostic tools related to acidosis is not straightforward, and that application of urine markers such as ammonium may require more nuance than would be predicted based on our understanding of the pathophysiology.
- Emerging Features of Ammonia Metabolism and Transport in Acid-Base Balance. [Review]
- SNSemin Nephrol 2019; 39(4):394-405
- Ammonia metabolism has a critical role in acid-base homeostasis and in other cellular functions. Kidneys have a central role in bicarbonate generation, which occurs through the process of net acid ex…
Ammonia metabolism has a critical role in acid-base homeostasis and in other cellular functions. Kidneys have a central role in bicarbonate generation, which occurs through the process of net acid excretion; ammonia metabolism is the quantitatively greatest component of net acid excretion, both under basal conditions and in response to acid-base disturbances. Several recent studies have advanced our understanding substantially of the molecular mechanisms and regulation of ammonia metabolism. First, the previous paradigm that ammonia transport could be explained by passive NH3 diffusion and NH4+ trapping has been advanced by the recognition that specific transport of NH3 and of NH4+ by specific membrane proteins is critical to ammonia transport. Second, significant advances have been made in the understanding of the regulation of ammonia metabolism. Novel studies have shown that hyperkalemia directly inhibits ammonia metabolism, thereby leading to the metabolic acidosis present in type IV renal tubular acidosis. Other studies have shown that the proximal tubule protein NBCe1, specifically the A variant NBCe1-A, has a major role in regulating renal ammonia metabolism. Third, there are important sex differences in ammonia metabolism that involve structural and functional differences in the kidney. This review addresses these important aspects of ammonia metabolism and transport.
- Intercalated Cells of the Kidney Collecting Duct in Kidney Physiology. [Review]
- SNSemin Nephrol 2019; 39(4):353-367
- The epithelium of the kidney collecting duct (CD) is composed mainly of two different types of cells with distinct and complementary functions. CD principal cells traditionally have been considered t…
The epithelium of the kidney collecting duct (CD) is composed mainly of two different types of cells with distinct and complementary functions. CD principal cells traditionally have been considered to have a major role in Na+ and water regulation, while intercalated cells (ICs) were thought to largely modulate acid-base homeostasis. In recent years, our understanding of IC function has improved significantly owing to new research findings. Thus, we now have a new model for CD transport that integrates mechanisms of salt and water reabsorption, K+ homeostasis, and acid-base status between principal cells and ICs. There are three main types of ICs (type A, type B, and non-A, non-B), which first appear in the late distal convoluted tubule or in the connecting segment in a species-dependent manner. ICs can be detected in CD from cortex to the initial part of the inner medulla, although some transport proteins that are key components of ICs also are present in medullary CD, cells considered inner medullary. Of the three types of ICs, each has a distinct morphology and expresses different complements of membrane transport proteins that translate into very different functions in homeostasis and contributions to CD luminal pro-urine composition. This review includes recent discoveries in IC intracellular and paracrine signaling that contributes to acid-base regulation as well as Na+, Cl-, K+, and Ca2+ homeostasis. Thus, these new findings highlight the potential role of ICs as targets for potential hypertension treatments.
- Effects of short-term fasting and different overfeeding diets on thyroid hormones in healthy humans. [Journal Article]
- TThyroid 2019 Jul 12
- Background Greater decrease in 24-h energy expenditure (EE) during fasting and smaller increase in 24-h EE during low-protein overfeeding (metabolic "thrifty" phenotype) predict weight gain. As thyro…
Background Greater decrease in 24-h energy expenditure (EE) during fasting and smaller increase in 24-h EE during low-protein overfeeding (metabolic "thrifty" phenotype) predict weight gain. As thyroid hormones (TH) are implicated in energy intake and metabolism, we assessed whether: 1) TH concentrations are altered by 24-h fasting or overfeeding diets with varying protein content; 2) diet-related changes in TH correlate with concomitant changes in EE. Methods Fifty-eight euthyroid, healthy subjects with normal glucose regulation underwent 24-h dietary interventions including fasting, eucaloric feeding, and five overfeeding diets in a crossover design within a whole-room indirect calorimeter to measure 24-h EE. Overfeeding diets (200% of energy requirements) included three diets with 20%-protein, one diet with 3%-protein (LPF: 46%-fat), and a diet with 30%-protein (HPF: 44%-fat, n=51). Plasma free thyroxine (FT4), triiodothyronine (FT3), and fibroblast growth factor 21 (FGF21) concentrations were measured after overnight fast the morning of and after each diet. Results On average, FT4 increased by 8% (+0.09 ng/dL, 95% CI: 0.06-0.13, p<0.001) while FT3 decreased by 6% (-0.17 pg/mL, CI: -0.27 to -0.07, p=0.001) after 24-h fasting, whereas both FT4 and FT3 decreased by 5% (-0.08 ng/dL, CI: -0.11 to -0.04, p<0.0001) and 4% (-0.14 pg/mL, CI: -0.24 to -0.04, p=0.008) following HPF, respectively. Greater decreases in FT3 after HPF associated with larger decreases in FGF21 (r=0.40, p=0.005). Following LPF, mean FT3 increased by 6% (+0.14 pg/mL, CI:0.05-0.2, p=0.003) with no change in FT4 (p=0.7). No changes in TH were observed after normal-protein overfeeding diets (all p>0.1). No associations were observed between TH concentrations and diet-related changes in 24-h EE during any diet (all p>0.07). Conclusion Acute (200%), short-term (24h) changes in food intake induce small changes in TH concentrations only after diets with low (0%-fasting and 3%-protein overfeeding) or high (30%-protein overfeeding) protein content. The FT3-FGF21 association after high-protein overfeeding suggests a role for TH in inhibiting FGF21 secretion by the liver during protein excess. These results indicate that TH are involved in protein metabolism; however, they do not mediate the short-term EE response to diets that characterize the metabolic phenotypes and determine the individual susceptibility to weight gain.
- A sparse covarying unit that describes healthy and impaired human gut microbiota development. [Journal Article]
- SciScience 2019 07 12; 365(6449)
- Characterizing the organization of the human gut microbiota is a formidable challenge given the number of possible interactions between its components. Using a statistical approach initially applied …
Characterizing the organization of the human gut microbiota is a formidable challenge given the number of possible interactions between its components. Using a statistical approach initially applied to financial markets, we measured temporally conserved covariance among bacterial taxa in the microbiota of healthy members of a Bangladeshi birth cohort sampled from 1 to 60 months of age. The results revealed an "ecogroup" of 15 covarying bacterial taxa that provide a concise description of microbiota development in healthy children from this and other low-income countries, and a means for monitoring community repair in undernourished children treated with therapeutic foods. Features of ecogroup population dynamics were recapitulated in gnotobiotic piglets as they transitioned from exclusive milk feeding to a fully weaned state consuming a representative Bangladeshi diet.
- Effects of microbiota-directed foods in gnotobiotic animals and undernourished children. [Journal Article]
- SciScience 2019 07 12; 365(6449)
- To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of…
To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.
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- Noninvasive In Vivo Quantification of Adeno-Associated Virus Serotype 9-Mediated Expression of the Sodium/Iodide Symporter Under Hindlimb Ischemia and Neuraminidase Desialylation in Skeletal Muscle Using Single-Photon Emission Computed Tomography/Computed Tomography. [Journal Article]
- CCCirc Cardiovasc Imaging 2019; 12(7):e009063
- CONCLUSIONS: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.