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Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5′→3′ exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information.

Significance In eukoryotes, 3′ to 5′ mRNA degradation is a major pathway to reduce mRNA levels and, thus, an important means to regulate gene expression. Herein, messenger RNA (mRNA) is hydrolyzed from the 3′ end by the exosome complex, producing short capped RNA fragments, which are decapped by DcpS. Our data show that DcpS is only active on mRNA that have undergone prior processing by the exosome. This DcpS selection mechanism is conserved from yeast to humans and is caused by the inability of the enzyme to undergo structural changes that are required for the formation of a catalytically active state around long mRNA transcripts. Our work thus reveals the mechanistic basis that ensures an efficient interplay between DcpS and the exosome., The 5′ messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3′ end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3′ to 5′ degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.

Mycobacterium chelonae is a ubiquitous Gram-positive, acid-fast, non-spore-forming bacterium commonly encountered in nature associated with aquatic animals, soil, and water, including tap water. Nontuberculous mycobacterial tenosynovitis infections caused by M. chelonae in the lower extremity are uncommon, leading to a paucity of literature documenting the diagnosis and treatment of such cases. This report is of a 65-year-old male patient who was found to have an M. chelonae infection along the tibialis anterior tendon after injecting himself with heroin into the dorsal foot veins. This review covers the diagnosis and treatment as well as a case report on the outcome of infectious tenosynovitis of the tibialis anterior associated with M. chelonae. To date, this is the only reported case of tibialis anterior infectious tenosynovitis caused by M. chelonae after intravenous heroin injection.

Background/Objective: Calcium loading has been associated with cardiovascular risk in hemodialysis (HD) patients. However, it remains to be elucidated whether alterations of intradialytic calcium buffering add to the increased cardiovascular disease burden in this high-risk population. Methods: Intradialytic calcium kinetics was evaluated in a cross-sectional observational study by measuring dialysate-sided ionized calcium mass balance (iCaMB), calcium buffer capacity, and change in serum calcium levels in 40 chronic HD patients during a routine HD session. A dialysate calcium of 3.5 mEq/L was used to adequately challenge calcium buffer mechanisms. Aortic pulse wave velocity and serum osteocalcin levels were measured prior to the HD session. Presence of cardiovascular disease and diabetes was assessed. Results: The mean dialysate-sided iCaMB, extracellular fluid ionized calcium mass gain, and buffered ionized calcium mass were 469 (±154), 111 (±49), and 358 (±145) mg/HD, respectively. The mean ionized serum calcium increase (∆iCa) was 0.42 (±0.14) mEq/L per HD. The mean intradialytic calcium buffer capacity was 73 (±18)%. Multivariate regression analysis revealed significant independent association of (1) iCaMB with the dialysate-to-blood calcium gradient at HD start and (2) intradialytic calcium buffer capacity with undercarboxylated osteocalcin. The presence of coronary heart disease was associated with higher ∆iCa but not iCaMB in the multivariate model. Conclusions: In line with our proof-of-concept study, we provide clinical evidence for a rapidly accessible and exchangeable calcium pool involved in intradialytic calcium regulation and for the role of osteocalcin as a potential biomarker. Our findings argue for evaluating the prognostic potential of intradialytic calcium kinetics in prospective clinical trials.

For the design of mitigation measures knowledge of debris-flow impact forces, usually estimated based on hydrostatic, hydrodynamic, or combined approaches, is essential. As these approaches are based on Newtonian fluids, they must be adjusted by empirical correction factors to account for the solid-fluid nature of debris flows. The values for the correction factors shown in the literature vary over a wide range and several studies showed a clear dependence with the Froude regime of debris flows.To better understand the correction factors and to be able to calculate them using parameters that describe the flow behaviour a total of 32 experiments were conducted in the course of the project “Debris flow impact forces on bridge super structures (DEFSUP)”, funded by the Austrian Science Fund (FWF). Two different material compositions, different water contents as well as a total impact and a bypassing of the measuring block were tested.The experimental setup designed within the project consists of a 4 m long semi-circular channel with a diameter of 300 mm and an inclination of 20°. The material is released from a rectangular reservoir in a dam-break scenario and accelerated with zero roughness on a length of 1.2 m and transferred to the semi-circle profile. The subsequently introduced roughness with a grain diameter of 1-2 mm generates a stationary phenomenological debris flow until it hits the measuring setup. With a starting volume of 50 kg, flow heights between 8 and 12 cm and velocities from 0.8 to 2.2 m/s were achieved according to the material composition and different water content. With these different mixtures a Froude-range from 0.6 to 3.6 was covered. In addition, normal stresses and pore water pressures were measured at the exact same point.A detailed analysis of the measured impact forces together with the above mentioned measured parameters showed that the hydrodynamic correction factor is a constant mainly corresponding to the liquification ratio of the debris-flow mixture. Hence, the hydrodynamic correction factor can be regarded as a drag coefficient and seems to depend mainly on the internal friction of the flowing medium. At low Froude numbers measured impact forces exceed even a full momentum transfer if the mean bulk density is used for the calculation. This indicates that the impact forces can no longer be described by the hydrodynamic approach alone. For this reason, an additional pressure term based on a hydrostatic approach is considered in the combined concept. This additional pressure term depends on the dynamics of flow (Froude number) and can be modelled via a dynamic earth pressure coefficient.The findings from these experiments contribute to a better prediction of debris-flows impact forces in terms of their material composition and flow behaviour.

SGLT2 inhibitors (SGLT2i) slow the progression of chronic kidney disease, however, evidence for the underlying molecular mechanisms is scarce. We investigated SGLT2i-mediated effects on differential gene expression in two independent human proximal tubular cell (HPTC) lines (HK-2 and RPTEC/TERT1) at the mRNA and protein levels under normoglycemic conditions, utilizing IL-1&beta, as a pro-inflammatory mediator. Microarray hybridization identified 259 genes that were uniformly upregulated by IL-1&beta, (10 mg/mL) and downregulated by empagliflozin (Empa) (500 nM) after 24 h of stimulation in two independent HPTC lines (n = 2, each). The functional annotation of these genes identified eight pathway clusters. Among 12 genes annotated to the highest ranked cluster (enrichment score, 3.51), monocyte chemoattractant protein-1/CC-chemokine ligand 2 (MCP-1/CCL2) and endothelin-1 (ET-1) were selected for verification at mRNA and protein levels based on their established involvement in the early pathogenesis of chronic kidney disease: IL-1&beta, upregulated basal MCP-1/CCL2 (15- and 19-fold) and ET-1 (3- and 8-fold) mRNA expression, while Empa downregulated basal MCP-1/CCL2 (0.6- and 0.5-fold) and ET-1 (0.3- and 0.2-fold) mRNA expression as early as 1 h after stimulation and for at least 24 h in HK-2 and RPTEC/TERT1 cells, respectively. The co-administration of Empa inhibited IL-1&beta, mediated MCP-1/CCL2 (0.2-fold, each) and ET-1 (0.2-fold, each) mRNA expression as early as 1 h after ligand stimulation and for at least 24 h in both HPTC lines, respectively. This inhibitory effect of Empa on basal and IL-1&beta, mediated MCP-1/CCL2 and ET-1 mRNA expression was corroborated at the protein level. Our study presents novel evidence for the interference of SGLT2 inhibition with tubular inflammatory response mechanisms under normoglycemic conditions that might account for SGLT2i-mediated nephroprotection.

The eukaryotic mRNA 5′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and high-resolution NMR spectroscopy. In combination with 13C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

Large clinical trials demonstrated that SGLT2 inhibitors (SGLT2i) slow the progression of kidney function decline in type 2 diabetes. Because the underlying molecular mechanisms are largely unknown, we studied the effects of SGLT2i on gene expression in two human proximal tubular (PT) cell lines under normoglycemic conditions, utilizing two SGLT2i, namely empagliflocin and canagliflocin. Genome-wide expression analysis did not reveal substantial differences between these two SGLT2i. Microarray hybridization analysis identified 94 genes that were both upregulated by TGF-β1 and downregulated by either of the two SGLT2i in HK-2 and RPTEC/TERT1 (renal proximal tubular epithelial cells/telomerase reverse transcriptase 1) cells. Extracellular matrix organization showed the highest significance in pathway enrichment analysis. Differential gene expression of three annotated genes of interest within this pathway was verified on mRNA level in both cell lines. Whereas TGF-β1 induced mRNA expression of thrombospondin 1 (THBS1; 4.3-fold), tenascin C (TNC; 8-fold), and platelet-derived growth factor subunit B (PDGF-B; 4.2-fold), SGLT2i downregulated basal mRNA expression of THBS1 (0.2-fold), TNC (0.5 fold), and PDGF-B (0.6-fold). Administration of SGLT2i in the presence of TGF-β1 resulted in a significant inhibition of TGF-β1-induced THBS1 and TNC mRNA expression and TGF-β1-induced THBS1, TNC, and PDGF-BB protein expression. We conclude that SGLT2i block basal and TGF-β1-induced expression of key mediators of renal fibrosis and kidney disease progression in two independent human PT cell lines.

Extraskeletal chondroma is a benign tumor that is found most often in the fingers but can be found in the feet as well. A symptom of this lesion is pressure from the slow-growing mass. We present the case of a 58-year-old woman who presented with an extraskeletal chondroma in the plantar aspect of the left first metatarsophalangeal joint with concomitant symptomatic arthrosis at the joint. Operative treatment was excision of the lesion in addition to arthrodesis of the joint attributable to the presence of symptomatic arthrosis. The patient was seen approximately 1 year postoperatively and had no postoperative complications. Distinction between extraskeletal chondromas and other lesions, such as extraskeletal myxoid chondrosarcomas, is critical because delayed treatment of the latter has the propensity to lead to detriment to the patient. Therefore, proper diagnosis is critical.

The scavenger decapping enzyme hydrolyzes the protective 5' cap structure on short mRNA fragments that are generated from the exosomal degradation of mRNAs. From static crystal structures and NMR data, it is apparent that the dimeric enzyme has to undergo large structural changes to bind its substrate in a catalytically competent conformation. Here we studied the yeast enzyme and showed that the associated opening and closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by the binding of a second ligand to the enzyme, which occurs at high substrate concentrations. We designed a mutant that disrupted the allosteric pathway that links the second binding event to the dynamics and showed that this mutant enzyme is hyperactive. Our data reveal a unique mechanism of substrate inhibition in which motions that are required for catalytic activity also inhibit efficient turnover when they are present in excess.