Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1).

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Shoulder magnetic resonance arthrography in dogs: Comparison of craniolateral and caudolateral approach for ultrasound-guided contrast injection.

Shoulder joint disease is a common cause of forelimb lameness in dogs. To diagnose this condition, shoulder magnetic resonance arthrography (MRA) is performed, which involves the injection of contrast agents into the shoulder joint space under ultrasound (US)-guidance. The objective of this study was to compare the craniolateral and caudolateral approaches for shoulder MRA using US-guided injection techniques, and investigate their clinical feasibility in dogs. Forty shoulder joints from 10 adult beagles were studied in two repetitions. The craniolateral (n = 20) and caudolateral (n = 20) injection techniques were applied randomly under US-guidance. The shoulder MRA was conducted immediately after the contrast agents was injected. The procedure time (scan and injection time), number of attempts, joint distension and degree of extraarticular extravasation were recorded and compared between the two groups. The results showed that the caudolateral approach had significantly more contrast agents extravasation compared to the craniolateral approach (p < 0.05). However, there were no significant differences between the two groups in terms of procedure time (scan time p = 0.80, injection time p = 0.74), number of attempts (p = 0.70) and joint distension (p = 0.23). The craniolateral approach of US-guided contrast injection techniques for shoulder MRA minimizes damage to the juxta-articular structures and reduces extraarticular extravasation, resulting in good-quality images. This study demonstrates the feasibility and advantages of the craniolateral approach under US-guidance for shoulder MRA in dogs.

Dipolar Noise in Fluorinated Molecular Wires.

We demonstrate a strategy to directly map and quantify the effects of dipole formation on electrical transports and noises in the self-assembled monolayers (SAMs) of molecular wires. In this method, the SAM patterns of fluorinated molecules with dipole moments were prepared on conducting substrates, and a conducting probe in contact-mode atomic force microscopy was utilized to map currents and noises through the probe on the molecular patterns. The maps were analyzed to extract the characteristic parameters of dipolar noises in SAMs, and the results were compared with those of hydrogenated molecular patterns without dipole moments. At rather low bias conditions, the fluorinated molecular junctions exhibited a tunneling conduction and a resistance value comparable to that of the hydrogenated molecules with a six-times-longer length, which was attributed to stronger dipoles formation in fluorinated molecules. Interestingly, conductance (G) in different regions of fluorinated molecular patterns exhibited a strong correlation with a noise power spectral density of SI/I2 like SI/I2 ∝ G-2, which can be explained by enhanced barrier fluctuations produced by the dipoles of fluorinated molecules. Furthermore, we observed that the noise power spectral density of fluorinated molecules showed an anomalous frequency (f) dependence like SI/I2 ∝ 1/f1.7, possibly due to the slowing down of the tunneling of carriers from increased barrier fluctuations. In rather high bias conditions, conductions in both hydrogenated and fluorinated molecules showed a transition from tunneling to thermionic charge transports. Our results provide important insights into the effects of dipoles on mesoscopic transport and resistance-fluctuation in molecules and could have a significant impact on the fundamental understanding and applications in this area.

Cryo-EM structures of GroEL:ES2 with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

The GroEL/GroES chaperonin system assists the folding of many proteins, through conformational transitions driven by ATP hydrolysis. Although structural information about bullet-shaped GroEL:ES1 complexes has been extensively reported, the substrate interactions of another functional complex, the football-shaped GroEL:ES2, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES2 complexes with a chemically denatured substrate, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Our structures demonstrate that native-like folded RuBisCO density is captured at the lower part of the GroEL chamber and that GroEL's bulky hydrophobic residues Phe281, Tyr360, and Phe44 contribute to direct contact with RuBisCO density. In addition, our analysis found that GroEL:ES2 can be occupied by two substrates simultaneously, one in each chamber. Together, these observations provide insights to the football-shaped GroEL:ES2 complex as a functional state to assist the substrate folding with visualization.

Feasibility and Reliability of Two-Dimensional Shear-Wave Elastography of the Liver of Clinically Healthy Cats.

Given the broad overlap of normal and abnormal liver tissue in the subjective evaluation of the liver in conventional B-mode ultrasonography, there is a need for a non-invasive and quantitative method for the diagnosis of liver disease. Novel two-dimensional shear-wave elastography (2-D SWE) can measure tissue stiffness by propagation of the shear wave induced using acoustic radiation force impulse in real time. To the best of our knowledge, two-dimensional shear-wave measurement of the liver in cats has not been reported to date. This study assessed the feasibility, reliability, normal values, and related influencing factors of 2-D SWE for assessment of the feline liver without anesthesia and breath-holding. Two-dimensional shear-wave ultrasonography was performed by two evaluators at the right and left sides of the liver. Twenty-nine client-owned clinically healthy adult cats were included. The means and standard deviations for the shear-wave speed and stiffness in the right liver were 1.52 ± 0.13 m/s and 6.94 ± 1.26 kPa, respectively, and those for the left liver were 1.61 ± 0.15 m/s and 7.90 ± 1.47 kPa, respectively. Shear-wave speed (P = 0.005) and stiffness (P = 0.002) were significantly lower in the right liver when compared to the left. The intraclass correlation value for liver stiffness was 0.835 and 0.901 for the right and left liver, respectively, indicating high interobserver agreement. Age, weight, body condition score (BCS), gabapentin administration, and measurement depths were not significantly correlated with liver stiffness or elastography measurements (P > 0.05). Our findings suggest that 2-D SWE measurements of the liver are not influenced significantly by age, weight, or BCS and can be reliably performed without anesthesia and breath-holding in cats. The values determined here can help form the basis for reference elastography values for evaluation of the feline liver.

SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions.

SIRT3 (sirtuin 3), a mitochondrial protein deacetylase, maintains respiratory function, but its role in the regulation of innate immune defense is largely unknown. Herein, we show that SIRT3 coordinates mitochondrial function and macroautophagy/autophagy activation to promote anti-mycobacterial responses through PPARA (peroxisome proliferator activated receptor alpha). SIRT3 deficiency enhanced inflammatory responses and mitochondrial dysfunction, leading to defective host defense and pathological inflammation during mycobacterial infection. Antibody-mediated depletion of polymorphonuclear neutrophils significantly increased protection against mycobacterial infection in sirt3-/- mice. In addition, mitochondrial oxidative stress promoted excessive inflammation induced by Mycobacterium tuberculosis infection in sirt3-/- macrophages. Notably, SIRT3 was essential for the enhancement of PPARA, a key regulator of mitochondrial homeostasis and autophagy activation in the context of infection. Importantly, overexpression of either PPARA or TFEB (transcription factor EB) in sirt3-/- macrophages recovered antimicrobial activity through autophagy activation. Furthermore, pharmacological activation of SIRT3 enhanced antibacterial autophagy and functional mitochondrial pools during mycobacterial infection. Finally, the levels of SIRT3 and PPARA were downregulated and inversely correlated with TNF (tumor necrosis factor) levels in peripheral blood mononuclear cells from tuberculosis patients. Collectively, these data demonstrate a previously unappreciated function of SIRT3 in orchestrating mitochondrial and autophagic functions to promote antimycobacterial responses. Abbreviations: Ab: antibody; BCG: M. bovis Bacillus Calmette-Guérin; Baf-A1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; CFU: colony forming unit; CXCL5: C-X-C motif chemokine ligand 5; EGFP: enhanced green fluorescent protein; ERFP: enhanced red fluorescent protein; FOXO3: forkhead box O3; HC: healthy controls; H&E: haematoxylin and eosin; HKL: honokiol; IHC: immunohistochemistry; IL1B: interleukin 1 beta; IL6: interleukin 6; IL12B: interleukin 12B; MDMs: monocyte-derived macrophages; MMP: mitochondrial membrane potential; Mtb: Mycobacterium tuberculosis; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PMN: polymorphonuclear neutrophil; PPARA: peroxisome proliferator activated receptor alpha; ROS: reactive oxygen species; SIRT3: sirtuin 3; TB: tuberculosis; TEM: transmission electron microscopy; TFEB: transcription factor EB; TNF: tumor necrosis factor.

PPAR-α Activation Mediates Innate Host Defense through Induction of TFEB and Lipid Catabolism.

The role of peroxisome proliferator-activated receptor α (PPAR-α) in innate host defense is largely unknown. In this study, we show that PPAR-α is essential for antimycobacterial responses via activation of transcription factor EB (TFEB) transcription and inhibition of lipid body formation. PPAR-α deficiency resulted in an increased bacterial load and exaggerated inflammatory responses during mycobacterial infection. PPAR-α agonists promoted autophagy, lysosomal biogenesis, phagosomal maturation, and antimicrobial defense against Mycobacterium tuberculosis or M. bovis bacillus Calmette-Guérin. PPAR-α agonists regulated multiple genes involved in autophagy and lysosomal biogenesis, including Lamp2, Rab7, and Tfeb in bone marrow-derived macrophages. Silencing of TFEB reduced phagosomal maturation and antimicrobial responses, but increased macrophage inflammatory responses during mycobacterial infection. Moreover, PPAR-α activation promoted lipid catabolism and fatty acid ß-oxidation in macrophages during mycobacterial infection. Taken together, our data indicate that PPAR-α mediates antimicrobial responses to mycobacterial infection by inducing TFEB and lipid catabolism.

Mycobacterium abscessus ESX-3 plays an important role in host inflammatory and pathological responses during infection.

Mycobacterial ESX systems are often related to pathogenesis during infection. However, little is known about the function of ESX systems of Mycobacterium abscessus (Mab). This study focuses on the Mab ESX-3 cluster, which contains major genes such as esxH (Rv0288, low molecular weight protein antigen 7; CFP-7) and esxG (Rv0287, ESAT-6 like protein). An esx-3 (MAB 2224c-2234c)-deletional mutant of Mab (Δesx) was constructed and used to infect murine and human macrophages. We then investigated whether Mab Δesx modulated innate host immune responses in macrophages. Mab Δesx infection resulted in less pathological and inflammatory responses. Additionally, Δesx resulted in significantly decreased activation of inflammatory signaling and cytokine production in macrophages compared to WT. Moreover, recombinant EsxG·EsxH (rEsxGH) proteins encoded by the ESX-3 region showed synergistic enhancement of inflammatory cytokine generation in macrophages infected with Δesx. Taken together, our data suggest that Mab ESX-3 plays an important role in inflammatory and pathological responses during Mab infection.

Upregulation of RSPO2-GPR48/LGR4 signaling in papillary thyroid carcinoma contributes to tumor progression.

The signaling pathway involving the R-spondins and its cognate receptor, GPR48/LGR4, is crucial in development and carcinogenesis. However, the functional implications of the R-spondin-GPR48/LGR4 pathway in thyroid remain to be identified. The aim of this study was to investigate the role of R-spondin-GPR48/LGR4 signaling in papillary thyroid carcinomas. We retrospectively reviewed a total of 214 patients who underwent total thyroidectomy and cervical lymph node dissection for papillary thyroid carcinoma. The role of GPR48/LGR4 in proliferation and migration was examined in thyroid cancer cell lines. R-spondin 2, and GPR48/LGR4 were expressed at significantly higher levels in thyroid cancer than in normal controls. Elevated GPR48/LGR4 expression was significantly associated with tumor size (P=0.049), lymph node metastasis (P=0.004), recurrence (P=0.037), and the BRAFV600E mutation (P=0.003). Moreover, high GPR48/LGR4 expression was an independent risk factor for lymph node metastasis (P=0.027) and the BRAFV600E mutation (P=0.009). in vitro assays demonstrated that elevated expression of GPR48/LGR4 promoted proliferation and migration of thyroid cancer cells, whereas downregulation of GPR48/LGR4 decreased proliferation and migration by inhibition of the ß-catenin pathway. Moreover, treatment of thyroid cancer cells with exogenous R-spondin 2 induced activation of the ß-catenin pathway through GPR48/LGR4. The R-spondin 2-GPR48/LGR4 signaling axis also induced the phosphorylation of ERK, as well as phosphorylation of LRP6 and serine 9 of GSK3ß. Our findings demonstrate that upregulation of the R-spondin 2-GPR48/LGR4 pathway contributes to tumor aggressiveness in papillary thyroid carcinoma by promoting ERK phosphorylation, suggesting that this pathway represents a novel therapeutic target for treatment of differentiated thyroid cancer.