Development of dynamic nomogram for predicting cancer-specific survival in hepatoid adenocarcinoma: A comprehensive SEER-based population analysis.

Hepatoid adenocarcinoma (HAC) is a poorly differentiated extrahepatic tumor that can produce alpha-fetoprotein (AFP). The literature does not provide a comprehensive understanding of the prognostic factors for HAC. Therefore, we present a novel nomogram to predict the cancer-specific survival (CSS) of patients with HAC. We analyzed 265 cases of HAC from the Surveillance, Epidemiology, and End Results (SEER) database spanning from 2004 to 2015. Using a Cox proportional hazard regression model, we identified several risk factors and incorporated them into our predictive nomogram. The nomogram's predictive ability was assessed by utilizing the concordance index (C-index), calibration curve, and receiver operating characteristic (ROC). Results from a multivariate Cox regression showed that CSS was independently correlated with liver metastasis, surgery, and chemotherapy. Our nomogram had a C-index of 0.71 (95% CI 0.71-0.96). Furthermore, calibration curves demonstrated concordance between the predicted survival probability from the nomogram and the observed survival probability. The areas under the curve (AUC) for 6-month, 1-, and 3-year survival were 0.80, 0.82, and 0.88, respectively. Our study successfully formulated a prognostic nomogram that offers promising predictions for the 6-month, 1-, and 3-year CSS of patients with HAC. This nomogram holds potential for practical use in guiding treatment decisions and designing clinical trials.

SENP1 attenuates hypoxia­reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF­1α signaling pathway.

Liver sinusoidal endothelial cells (LSECs) have an important role in hepatic ischemia­reperfusion injury (I/R), but the specific molecular mechanism of action is unknown. LSEC proliferation is regulated and fenestration is maintained via the Sentrin/SUMO­specific protease 1 (SENP1)/hypoxia­inducible factor­1α (HIF­1α) signaling axis under hypoxic conditions. In the present study, a hypoxia­reoxygenation (H­R) injury model was established using mouse LSECs to explore the relationship between SENP1 and H­R injury in vitro, and the specific underlying mechanism was identified, revealing new targets for the clinical attenuation of hepatic I/R injury. Following the culture of LSECs under H­R conditions, it was demonstrated that the expression of SENP1 was upregulated by reverse transcription­quantitative polymerase chain reaction and western blotting (WB). In addition, scanning electron microscopy indicated that fenestrae damage was increased, a Cell Counting Kit­8 assay demonstrated that the proliferation of cells was impaired and flow cytometry showed that apoptosis was increased. After silencing SENP1 expression with short interfering RNA, the proliferation activity of LSECs decreased, the fenestrae damage increased, the apoptosis rate increased and the expression levels of SENP1, HIF­1α, heme oxygenase and Bcl­2 were downregulated (as demonstrated by WB), while the expression levels of apoptosis­related proteins, cleaved­caspase­3 and Bax, were upregulated. Enzyme­linked immunosorbent assay detection showed that the level of vascular endothelial growth factor in the supernatant decreased and the level of IL­6 and TNF­α increased. Following the administration of an HIF­1α signaling pathway agonist, the situation was reversed. These results therefore suggested that SENP1 attenuated the reduction in proliferation, apoptosis and fenestration of LSECs observed following H­R injury through the HIF­1α signaling pathway. In conclusion, SENP1 may attenuate H­R injury in LSECs in a HIF­1α signaling pathway­dependent manner.

Biomimetic pheomelanin to unravel the electronic, molecular and supramolecular structure of the natural product.

Herein, we investigate synthetic routes to a close mimic of natural pheomelanin. Three different oxidative polymerization routes were attempted to generate synthetic pheomelanin, each giving rise to structurally dissimilar materials. Among them, the route employing 5-cysteinyl-dihydroxyphenylalanine (5-CD) as a monomer was verified as a close analogue of extracted pheomelanin from humans and birds. The resulting biomimetic and natural pheomelanins were compared via various techniques, including solid-state Nuclear Magnetic Resonance (ssNMR) and Electron Paramagnetic Resonance (EPR). This synthetic pheomelanin closely mimics the structure of natural pheomelanin as determined by parallel characterization of pheomelanin extracted from multiple biological sources. With a good synthetic biomimetic material in hand, we describe cation-π interactions as an important driving force for pheomelanogenesis, further advancing our fundamental understanding of this important biological pigment.

Ginsenoside Rg1 Alleviates Rat Liver Ischemia-Reperfusion Ischemia Through Mitochondrial Autophagy Pathway.

Aim: The aim of this study was to elucidate the potential mechanism of Rg1 in alleviating hepatic ischemia-reperfusion (HIRI) through the mitophagy pathway. Methods: The HIRI rat models were established and divided into 4 groups: the sham group, sham+Rg1 group, ischemia/perfusion (I/R) group and I/R+Rg1 group. Then the activities of aspartate transaminase (AST) and alanine aminotransferase (ALT) were detected by automatic serum analyzer. Meanwhile, cell apoptosis and changes in liver tissues were checked by TUNEL assay and histopathological analysis, respectively. The relative protein levels were detected by western blotting. Subsequently, cell counting Kit-8 assay and cytometric analysis were used to investigate cell viability and apoptosis of liver cells. Finally, the time points of the strongest mitochondrial autophagy were explored and the mitochondrial morphology was observed by the mitochondrial transmembrane potential (MMP) in vivo and in vitro. Results: The mitophagy aggravated hepatocyte damage during liver I/R in vivo. In addition, Rg1 alleviated liver damage after liver I/R, maintained the stability of MMP and inhibited mitochondrial autophagy and signaling pathways during liver I/R in vivo. Furthermore, Rg1 could effectively increase cell viability, inhibit cell apoptosis and stabilize MMP after OGD/R injury in vitro Moreover, Rg1 exerted its protective effect on HIRI by regulating the PINK1/Parkin signaling pathway and the mitochondrial autophagy. Conclusion: Rg1 could further improve its mechanism of alleviating HIRI in apoptosis and autophagy, 2 types of regulated programmed cell death via the mitochondrial pathway.

Chemoenzymatic Isolation and Characterization of High Purity Mammalian Melanin.

Although melanin is one of the most ubiquitous polymers in living systems, our understanding of its molecular structure, biosynthesis and biophysical properties has been limited to only a small number of organisms other than humans. This is in part due to the difficulty associated with isolating pure melanin. While purification methods exist, they typically involve harsh treatments with strong acid/base conditions combined with elevated temperatures that can lead to the polymer backbone degradation. To be successful, a viable isolation method must deliver a selective, yet complete degradation of non-melanin biopolymers as well as remove small molecule metabolites that are not integrative to the melanin backbone. Here, we demonstrate the use of chemoenzymatic processing guided by fluorescent probes for the purification and isolation of native mammalian melanin without significant induction of chemical degradation. This multi-step purification-tracking methodology enables quantitative isolation of pure melanin from mammalian tissue for spectroscopic characterization.

Caffeine Inhibits Activation of the NLRP3 Inflammasome via Autophagy to Attenuate Microglia-Mediated Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

The activation of microglia is an important cause of central nervous system (CNS) inflammatory cell infiltration and inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). Furthermore, the proinflammatory response induced by the NLR family pyrin domain containing 3 (NLRP3) inflammasome can be amplified in microglia after NLRP3 inflammasome activation. Autophagy is closely related to the inflammatory response. Caffeine exerts anti-inflammatory and autophagy-stimulating effects, but the specific mechanism remains unclear. This study examined the mechanism underlying the anti-inflammatory effect of caffeine on EAE. In this study, C57BL/6 mice were immunized to induce EAE and treated with caffeine to observe its effect on prognosis. The effects of caffeine on autophagy and inflammation were also analysed in mouse primary microglia (PM) and the BV2 cell line. The data demonstrated that caffeine reduced the clinical score, the infiltration of inflammatory cells, the demyelination level, and the activation of microglia in EAE mice. Furthermore, caffeine increased the LC3-II/LC3-I levels and decreased the NLRP3 and P62 levels in EAE mice, whereas the autophagy inhibitor 3-methylamine (3-MA) blocked these effects. In vitro, caffeine promoted autophagy by suppressing the mechanistic target of rapamycin (mTOR) pathway and inhibited activation of the NLRP3 inflammasome. However, autophagy-related gene 5 (ATG5)-specific siRNA abolished the anti-inflammatory effect of caffeine treatment in PM and BV2 cells. Taken together, these data suggest that caffeine exerts a newly discovered effect on EAE by reducing NLRP3 inflammasome activation via the induction of autophagy in microglia.

Hypoxia promotes the proliferation of mouse liver sinusoidal endothelial cells: miRNA-mRNA expression analysis.

During the initial stage of liver regeneration (LR), hepatocytes and liver sinusoidal endothelial cells (LSECs) initiate regeneration in a hypoxic environment. However, the role of LSECs in liver regeneration in hypoxic environments and their specific molecular mechanism is unknown. Therefore, this study aimed to explore the miRNA-mRNA network that regulates the proliferation of LSECs during hypoxia. In this study, first, we found that the proliferation ability of primary LSECs treated with hypoxia was enhanced compared with the control group, and then whole transcriptome sequencing was performed to screen 1837 differentially expressed (DE) genes and 17 DE miRNAs. Subsequently, the bioinformatics method was used to predict the target genes of miRNAs, and 309 pairs of interacting miRNA-mRNA pairs were obtained. Furthermore, the miRNA-gene action network was established using the negative interacting miRNA-mRNA pairs. The selected mRNAs were analyzed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and biological processes (BP) and signal pathways related to LSEC proliferation that were significantly enriched in GO-BP and KEGG were selected. Finally, 22 DE genes and 17 DE miRNAs were screened and the network was created. We also successfully verified the significant changes in the top six genes and miRNAs using qRT-PCR, and the results were consistent with the sequencing results. This study proposed that a specific miRNA-mRNA network is associated with hypoxia-induced proliferation of LSECs, which will assist in elucidating the potential mechanisms involved in hypoxia-promoting liver regeneration during LR.

Unraveling the Structure and Function of Melanin through Synthesis.

Melanin is ubiquitous in living organisms across different biological kingdoms of life, making it an important, natural biomaterial. Its presence in nature from microorganisms to higher animals and plants is attributed to the many functions of melanin, including pigmentation, radical scavenging, radiation protection, and thermal regulation. Generally, melanin is classified into five types-eumelanin, pheomelanin, neuromelanin, allomelanin, and pyomelanin-based on the various chemical precursors used in their biosynthesis. Despite its long history of study, the exact chemical makeup of melanin remains unclear, and it moreover has an inherent diversity and complexity of chemical structure, likely including many functions and properties that remain to be identified. Synthetic mimics have begun to play a broader role in unraveling structure and function relationships of natural melanins. In the past decade, polydopamine, which has served as the conventional form of synthetic eumelanin, has dominated the literature on melanin-based materials, while the synthetic analogues of other melanins have received far less attention. In this perspective, we will discuss the synthesis of melanin materials with a special focus beyond polydopamine. We will emphasize efforts to elucidate biosynthetic pathways and structural characterization approaches that can be harnessed to interrogate specific structure-function relationships, including electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. We believe that this timely Perspective will introduce this class of biopolymer to the broader chemistry community, where we hope to stimulate new opportunities in novel, melanin-based poly-functional synthetic materials.

Hypoxia maintains the fenestration of liver sinusoidal endothelial cells and promotes their proliferation through the SENP1/HIF-1α/VEGF signaling axis.

Liver sinusoidal endothelial cells (LSECs), which play a very critical role in liver regeneration, function in hypoxic environments, but few studies have elucidated the specific mechanism. As a hypoxia-sensitive gene, Sentrin/SUMO-specific protease 1(SENP1) is upregulated in solid tumors due to hypoxia and promotes tumor proliferation. We speculate that LSECs may upregulate SENP1 in hypoxic environments and that SENP1 may act on downstream genes to allow the cells to adapt to the hypoxic environment. To elucidate the reasons for the survival of LSECs under hypoxia, we designed experiments to explore the possible mechanism. First, we cultured murine LSECs in hypoxic conditions for a certain time (24 h and 72 h), and then, we observed that the proliferation ability of the hypoxia group was higher than that of the normoxia group, and the number of unique fenestrae of the LSECs in the hypoxia group was more than that of the LSECs in the normoxia group. Then, we divided the LSECs into several groups for hypoxic culture for time points (6 h, 12 h, 24 h, 36 h, and 72 h), and we found that the expression of SENP1, HIF-1α and VEGF was significantly upregulated. Then, we silenced SENP1 and HIF-1α with si-SENP1 and si-HIF-1α, respectively. SENP1, HIF-1α and VEGF were significantly downregulated, as determined by RT-PCR, WB and ELISA. Unexpectedly, the proliferation activity of the LSECs decreased and the fenestrae disappeared more in the si-SENP1 and si-HIF-1α groups than in the control group. It is concluded that LSECs cultured under hypoxic conditions may maintain fenestrae and promote proliferation through the SENP1/HIF-1α/VEGF signaling axis, thereby adapting to the hypoxic environment.

Isolation and Characterization of Allomelanin from Pathogenic Black Knot Fungus-a Sustainable Source of Melanin.

Melanin, a widespread pigment found in many taxa, is widely recognized for its high refractive index, ultraviolet (UV) protection, radical quenching ability, metal binding, and many other unique properties. The aforementioned characteristic traits make melanin a potential candidate for biomedical, separation, structural coloration, and space applications. However, the commercially available natural (sepia) and synthetic melanin are very expensive, limiting their use in various applications. Additionally, eumelanin has been the primary focus in most of these studies. In the present study, we demonstrate that melanin can be extracted from the pathogenic black knot fungus Apiosporina morbosa with a yield of ∼10% using the acid-base extraction method. The extracted melanin shows irregular morphology. Chemical characterization using X-ray photoelectron spectroscopy, infrared spectroscopy, and solid-state nuclear magnetic resonance spectroscopy reveals that the melanin derived from black knots is the less explored nitrogen-free allomelanin. Additionally, the extracted melanin shows broadband UV absorption typical of other types of melanin. Because of the wide availability and low cost of black knots and the invasive nature of the fungus, black knots can serve as an alternative green source for obtaining allomelanin at a low cost, which could stimulate its use as an UV light absorber and antioxidant in cosmetics and packaging industries.

Selenomelanin: An Abiotic Selenium Analogue of Pheomelanin.

Melanins are a family of heterogeneous biopolymers found ubiquitously across plant, animal, bacterial, and fungal kingdoms where they act variously as pigments and as radiation protection agents. There exist five multifunctional yet structurally and biosynthetically incompletely understood varieties of melanin: eumelanin, neuromelanin, pyomelanin, allomelanin, and pheomelanin. Although eumelanin and allomelanin have been the focus of most radiation protection studies to date, some research suggests that pheomelanin has a better absorption coefficient for X-rays than eumelanin. We reasoned that if a selenium enriched melanin existed, it would be a better X-ray protector than the sulfur-containing pheomelanin because the X-ray absorption coefficient is proportional to the fourth power of the atomic number (Z). Notably, selenium is an essential micronutrient, with the amino acid selenocysteine being genetically encoded in 25 natural human proteins. Therefore, we hypothesize that selenomelanin exists in nature, where it provides superior ionizing radiation protection to organisms compared to known melanins. Here we introduce this novel selenium analogue of pheomelanin through chemical and biosynthetic routes using selenocystine as a feedstock. The resulting selenomelanin is a structural mimic of pheomelanin. We found selenomelanin effectively prevented neonatal human epidermal keratinocytes (NHEK) from G2/M phase arrest under high-dose X-ray irradiation. Provocatively, this beneficial role of selenomelanin points to it as a sixth variety of yet to be discovered natural melanin.

MBNL1 reverses the proliferation defect of skeletal muscle satellite cells in myotonic dystrophy type 1 by inhibiting autophagy via the mTOR pathway.

Skeletal muscle atrophy is one of the clinical symptoms of myotonic dystrophy type 1 (DM1). A decline in skeletal muscle regeneration is an important contributor to muscle atrophy. Skeletal muscle satellite cells (SSCs) drive skeletal muscle regeneration. Increased autophagy can reduce the proliferative capacity of SSCs, which plays an important role in the early regeneration of damaged skeletal muscle in DM1. Discovering new ways to restore SSC proliferation may aid in the identification of new therapeutic targets for the treatment of skeletal muscle atrophy in DM1. In the pathogenesis of DM1, muscleblind-like 1 (MBNL1) protein is generally considered to form nuclear RNA foci and disturb the RNA-splicing function. However, the role of MBNL1 in SSC proliferation in DM1 has not been reported. In this study, we obtained SSCs differentiated from normal DM1-04-induced pluripotent stem cells (iPSCs), DM1-03 iPSCs, and DM1-13-3 iPSCs edited by transcription activator-like (TAL) effector nucleases (TALENs) targeting CTG repeats, and primary SSCs to study the pathogenesis of DM1. DM1 SSC lines and primary SSCs showed decreased MBNL1 expression and elevated autophagy levels. However, DM1 SSCs edited by TALENs showed increased cytoplasmic distribution of MBNL1, reduced levels of autophagy, increased levels of phosphorylated mammalian target of rapamycin (mTOR), and improved proliferation rates. In addition, we confirmed that after MBNL1 overexpression, the proliferative capability of DM1 SSCs and the level of phosphorylated mTOR were enhanced, while the autophagy levels were decreased. Our data also demonstrated that the proliferative capability of DM1 SSCs was enhanced after autophagy was inhibited by overexpressing mTOR. Finally, treatment with rapamycin (an mTOR inhibitor) was shown to abolish the increased proliferation capability of DM1 SSCs due to MBNL1 overexpression. Taken together, these data suggest that MBNL1 reverses the proliferation defect of SSCs in DM1 by inhibiting autophagy via the mTOR pathway.

Chemoenzymatic elaboration of the Raper-Mason pathway unravels the structural diversity within eumelanin pigments.

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved. Here, we apply a biosynthetic approach to explore the composite structures accessible in one type of melanin, eumelanin. Using a combination of solid-state NMR, dynamic nuclear polarization, and electron microscopy, we reveal how a variety of monomers are enzymatically polymerized into their corresponding eumelanin pigments. We demonstrate how this approach can be used to unite structure with an understanding of enzymatic activity, substrate scope, and the regulation of nanostructural features. Overall, this data reveals how intermediate metabolites of the Raper-Mason metabolic pathway contribute to polymerization, allowing us to revisit the original proposal of how eumelanin is biosynthesized.

NUF2 is a valuable prognostic biomarker to predict early recurrence of hepatocellular carcinoma after surgical resection.

Early tumor recurrence after curative surgical resection poses a great challenge to the clinical management of hepatocellular carcinoma (HCC). We conducted whole genome expression microarrays on 64 primary HCC tumors with clinically defined recurrence status and cross-referenced with RNA-seq data from 18 HCC tumors in the Cancer Genome Atlas project. We identified a 77-gene signature, which is significantly associated with early recurrent (ER) HCC tumors. This ER-associated signature shows significant enrichment in genes involved in cell cycle pathway. We performed receiver operating characteristic (ROC) analysis to evaluate the prognostic biomarker potential of these 77 genes and Pearson correlation analysis to identify 11 close clusters. The one gene with the best area under the ROC curve in each of the 11 clusters was selected for validation using reverse-transcription quantitative PCR in an independent cohort of 24 HCC tumors. NUF2 was identified to be the minimal biomarker sufficient to discriminate ER tumors from LR tumors. NUF2 in combination with liver cirrhosis could significantly improve the detection of ER tumors with an AUROC of 0.82 and 0.85 in the test and validation cohort, respectively. In conclusion, NUF2 in combination with liver cirrhosis is a promising prognostic biomarker for early HCC recurrence.

Order and disorder in (E)-[1-(biphenyl-4-yl)ethylidene]hydrazine: a structural, spectroscopic and theoretical study.

An unexpected global disorder (co-existing rotational disorder and glide disorder) has been observed during an X-ray investigation of the crystal structure of (E)-[1-(biphenyl-4-yl)ethylidene]hydrazine, C14H14N2, at room temperature. When the temperature decreases to 273 K, the disorder disappears, but the quality of the data set is low. The diffraction data were collected again at 110 K. Differential scanning calorimetry (DSC) analysis and polarizing-microscopy experiments, as well as a fourth set of single-crystal data collected at 283 K, proved that the order-disorder transformation occurs continuously. The analyses of these crystal structures and full-range relaxed potential energy surface scans showed that this kind of global disorder is not very difficult to achieve inside the crystal. Experimental and theoretical studies via UV-Vis and fluorescence spectra impart an understanding on the prediction methods of optical properties, which are essential for the rational design of biphenyl-based materials with pre-defined properties.

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora.

AIM: To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine. METHODS: Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro. RESULTS: TOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut. CONCLUSION: Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

In Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR.

A principal advantage of magic angle spinning (MAS) NMR spectroscopy lies in its ability to determine molecular structure in a noninvasive and quantitative manner. Accordingly, MAS should be widely applicable to studies of the structure of active pharmaceutical ingredients (API) and formulations. However, the low sensitivity encountered in spectroscopy of natural abundance APIs present at low concentration has limited the success of MAS experiments. Dynamic nuclear polarization (DNP) enhances NMR sensitivity and can be used to circumvent this problem provided that suitable paramagnetic polarizing agent can be incorporated into the system without altering the integrity of solid dosages. Here, we demonstrate that DNP polarizing agents can be added in situ during the preparation of amorphous solid dispersions (ASDs) via spray drying and hot-melt extrusion so that ASDs can be examined during drug development. Specifically, the dependence of DNP enhancement on sample composition, radical concentration, relaxation properties of the API and excipients, types of polarizing agents and proton density, has been thoroughly investigated. Optimal enhancement values are obtained from ASDs containing 1% w/w radical concentration. Both polarizing agents TOTAPOL and AMUPol provided reasonable enhancements. Partial deuteration of the excipient produced 3× higher enhancement values. With these parameters, an ASD containing posaconazole and vinyl acetate yields a 32-fold enhancement which presumably results in a reduction of NMR measurement time by ∼1000. This boost in signal intensity enables the full assignment of the natural abundance pharmaceutical formulation through multidimensional correlation experiments.

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR.

In DNP MAS NMR experiments at ∼80-110 K, the structurally important -13CH3 and -15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78-300 K. In APG, the three-site hopping of the Ala-Cß peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C's and 15N's. Similarly, the 15N signal from Ala-NH3+ disappears at ∼173 K, concurrent with the attenuation in CP experiments of other 15N's as well as 13C's. In bR and PI3-SH3, the methyl groups are attenuated at ∼95 K, while all other 13C's remain unaffected. However, both systems exhibit substantial losses of intensity at ∼243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

Endoscopic surgery of a sinonasal inverted papilloma: Surgical strategy, follow-up, and recurrence rate.

BACKGROUND: Sinonasal inverted papilloma (SNIP) is noted for its high rate of recurrence and malignant transformation. Although many clinical studies have demonstrated the effectiveness of the endoscopic approach for SNIP, the surgical strategy has been the subject of much debate. OBJECTIVE: To evaluate the effectiveness of the endoscopic endonasal approach in SNIP. METHODS: A systematic review of patients with a diagnosis of SNIP and who had surgery at our institution from June 2005 to March 2013 was performed. All the patients who had postoperative follow-up for >2 years were enrolled. Each case was categorized into one of four stages as reported by Krouse. Demographic and tumor date, operative approach, complications, and recurrence rates were collected. RESULTS: A total of 125 patients were included in this study. There were 17 patients in stage 1, 40 in stage 2, 57 in stage 3, and 11 in stage 4. The overall recurrence rate was 8.0%. There was no significant difference in recurrence among the stages (all p > 0.05). Recurrence after endoscopic endonasal approach (8.4%) and a combined endoscopic and open exposure procedure (5.6%) were not significantly different (p > 0.05). The recurrence rate was significantly (p < 0.05) higher in patients with revision (15.6%) than in patients in the primary cases (3.8%). A common site of tumor origin was recorded to be from the maxillary sinus (40.2%). Twenty percent of recurrences were observed up to 5 years after surgery. CONCLUSION: Endoscopic surgery may be preferred for treating SNIP. The elevated recurrence rate after revision emphasized the significance of the first surgery. We encourage a follow-up period of at least 5 years.