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BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5(+) cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Because of the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit medium, including DNA/RNA Shield ™, NAT, VTM-N, Ezmedlab™, and Phosphate Buffered Saline (PBS), for nasopharyngeal swab samples (NPSs) from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered a higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield ™ shows higher relative fluorescence units (RFU) and lower Cq values for the reference gene. Collectively, our results suggest that PBS medium could compromise the sample diagnosis because its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N and DNA/RNA Shield ™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis. This article is protected by copyright. All rights reserved.

The therapeutic potential of the Meitner-Auger- and conversion-electron emitting radionuclide 119Sb remains unexplored because of the difficulty of incorporating it into biologically targeted compounds. To address this challenge, we report the development of 119Sb production from electroplated tin cyclotron targets and its complexation by a novel trithiol chelate. The chelation reaction occurs in harsh solvent conditions even in the presence of large quantities of tin, which are necessary for production on small, low energy (16 MeV) cyclotrons. The 119Sb-trithiol complex has high stability and can be purified by HPLC. The third generation trithiol chelate and the analogous stable natSb-trithiol compound were synthesized and characterized, including by single-crystal X-ray diffraction analyses.

In animal tissues, N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactive N-acylethanolamines, in which an acyl chain is transferred from the sn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation of N-acylphosphatidylethanolamine. NAT has long been known to be stimulated by Ca(2+), and hence it has been referred to as Ca(2+)-dependent NAT. On the other hand, members of the phospholipase A/acyltransferase (PLA/AT) family (also known as HRAS-like suppressor family) show Ca(2+)-independent NAT activity. In this chapter, we describe (1) partial purification of Ca(2+)-dependent NAT from rat brain, (2) purification of recombinant PLA/AT-2, and (3) NAT assay using radiolabeled substrate.

The total synthesis of (+)-03219A, a rare Δ8,9-pregnene isolated from the marine-derived Streptomyces sp. SCSIO 03219, is described that is based on a series of transformations that enable progression from epichlorohydrin to an ent-estrane, then conversion to a nat-androstane, and finally establishment of the natural product target. Key to the success of these studies was implementation of two rearrangement processes to formally invert the quaternary center at C13 and establish the C10 quaternary center.

The early determination of response to neoadjuvant therapy (NAT) in triple-negative breast cancer would enable the treating oncologist to adapt the therapeutic regimen of a non-responding patient (e.g., by changing dosage, dose schedule, prescribed drugs), and thereby improve treatment outcomes while avoiding unnecessary toxicities. To address this challenge, we propose to use personalized, in silico forecasts of tumor response to therapeutic regimens via a mechanistic mathematical model calibrated with patient-specific longitudinal multi-parametric magnetic resonance imaging (MRI) data acquired early in the course of NAT. Here, we extend our mechanistic model to include a new term describing the synergistic effects of NAT drug combinations and identify the driving parameters involved in its formulation by means of a sensitivity analysis. Our model describes tumor cell dynamics as a combination of proliferation, which is regulated by a logistic term, and mobility, which is described as a diffusion process constrained by the local tumor-induced mechanical stress. Tumor cell density is extracted from diffusion-weighted MRI data, while tissue mechanical properties are defined from segmented T1-weighted MRI data. We adjust the tumor proliferation rate in response to NAT drug combinations with a recent model of drug synergy, MuSyC, which accounts for distinct types of synergistic drug effects (synergy of potency vs. synergy of efficacy). We also consider the heterogeneous intratumoral delivery of drugs by means of perfusion maps estimated from dynamic contrast-enhanced MRI data. We use Sobol’s method for the sensitivity analysis of two different tumors - one well-perfused and one poorly-perfused. We simulate a four-cycle NAT protocol in which NAT drugs are delivered every 14 days, and assess the total effect (ST) of each parameter on the mean relative difference of tumor cell density with respect to a control simulation of tumor growth without NAT. Sensitivity analysis results directly depend on the definition of the parameter space, which we construct by combining two approaches. First, we experimentally constrain parameter ranges using time-resolved, high-throughput, automated microscopy assays to capture the changes in proliferation rates of various breast cancer lines (HCC1143, SUM149, MDAMB231, and MDAMB468) caused by two standard drug combinations: paclitaxel with carboplatin and doxorubicin with perfosfamide (metabolic derivative of the pro-drug cyclophosphamide), and fitting the MuSyC model to these data. Second, we scale the resulting in vitro parameter ranges to clinically-relevant in vivo ranges by running an in silico study with our mechanistic model of breast cancer growth and NAT response. Our results show that, out of the ten parameters involved in the synergy term, three have a dominant role in the dynamics of breast cancer during NAT (ST > 0.1): synergistic potency, the maximal change in tumor cell proliferation by the slowest decaying drug, and its concentration producing half of maximal effects. The other parameters have marginal (0.02 < ST < 0.1) to negligible effect (ST < 0.02). Ongoing studies are assessing the ability of our mechanistic model to forecast NAT response over a small patient cohort after patient-specific calibration of the driving parameters identified in the present study. Citation Format: Guillermo Lorenzo, Angela M. Jarrett, Christian T. Meyer, Darren R. Tyson, Vito Quaranta, Thomas E. Yankeelov. Identifying relevant parameters that characterize the early response to NAT in breast cancer patients using a novel personalized mechanistic model integrating in vitro and in vivo imaging data [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-44.

Aim: Δ9-Tetrahydrocannabinol (Δ9-THC) is a potentially addictive cannabinoid. Its impact on the activity of liver arylamine N-Acetyltransferase (NAT) has not been reported. This study investigated the rewarding effects of Δ9-THC in mice and whether Δ9-THC had any impact ex-vivo and in-vitro on NAT activity. Methods: Thirty-six Swiss albinomice randomly assigned to six groups (n = 6) completed a biased, 8-week Conditioned Place Preference (CPP) paradigm. Mice exhibiting ~80% preference for the black chamber at pre-conditioning were selected. Treatment groups were administered Δ9-THC (0.10, 0.50 or 2.0 mg/kg/mL, ip) or amphetamine (AMP, 5.0 mg/kg/mL, ip); while untreated groups (controls) received vehicle solutions (coconut oil or 0.9% saline). Entries and time spent in the white, drug-paired chamber during a 15-min post-conditioning exploration of the CPP apparatus were compared with the pre-conditioning exploratory scores. Livers from Δ9-THC treated and untreated mice were excised and NAT enzyme activity determined ex-vivo using a spectrophotometric assay with p-anisidine as substrate. The impact of varying concentrations of Δ9-THC (0.00 - 162 μM) on the activities of NAT from untreated mice livers were also investigated in-vitro. Results: Δ9-THC treated mice entered and spent significantly more time in the drug-paired CPP chamber (p ≤ 0.05) at post-conditioning vs pre-conditioning (F = 11.22). Mice treated with 2.0 mg/kg Δ9-THC made significantly more entries into the drug-paired chamber (p ≤ 0.05) as compared with their vehicle controls. AMP-treated mice displayed significant (p In-vitro NAT evaluations revealed a dose-dependent inhibitory impact of Δ9-THC on NAT activity with an IC50 value of 34.97 ±1.58 μM. Ex-vivo evaluations of livers from Δ9-THC treated mice showed no significant impact on liver NAT enzyme activity. Conclusion: Δ9-THC induced significant conditioned place preference (drug reward) and produced a moderate dose-dependent inhibition on NAT activity in-vitro, but not ex-vivo.

The members of the arylamine N-acetyltransferase (NAT) family of enzymes are important for their many roles in xenobiotic detoxification in bacteria and humans. However, very little is known about their roles outside of detoxification or their specificities for acyl donors larger than acetyl-CoA. Herein, we report the detailed study of PtmC, an unusual NAT homologue encoded in the biosynthetic gene cluster for thioplatensimycin, thioplatencin, and a newly reported scaffold, thioplatensilin, thioacid-containing diterpenoids and highly potent inhibitors of bacterial and mammalian fatty acid synthases. As the final enzyme of the pathway, PtmC is responsible for the selection of a thioacid arylamine over its cognate carboxylic acid and coupling to at least three large, 17-carbon ketolide-CoA substrates. Therefore, this study uses a combined approach of enzymology and molecular modeling to reveal how PtmC has evolved from the canonical NAT scaffold into a key part of a natural combinatorial biosynthetic pathway. Additionally, genome mining has revealed the presence of other related NATs located within natural product biosynthetic gene clusters. Thus, findings from this study are expected to expand our knowledge of how enzymes evolve for expanded substrate diversity and enable additional predictions about the activities of NATs involved in natural product biosynthesis and xenobiotic detoxification.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. publishedVersion

The nanozyme concept appeals to numerous scientists with interest in cancer catalytic therapy based on an easy and economic preparation with highly catalytic and stable properties compared to a natural enzyme. The supramolecular nanozyme is an especially efficient and simple method, which has been successful in biomedical applications by changing the redox state and reactive oxygen species in a cancer environment. Representative examples of nanozymes and supramolecular nanozymes in cancer catalytic therapy with respect to construction and development are displayed in this review. Emphasis is also placed on the supramolecular nanozyme materials and the inherent structure of those containing nanoparticles, hydrogels, nanovesicles, and the catalytic mechanism and reaction. The supramolecular nanozyme, promising for its special properties of selectivity, high catalytic capability and stability, could reveal the mysterious properties of the enzyme itself, and the efficient inhibition of the signaling channel in cancer catalytic therapy.

Novel 2-amino(substituted pyrimidin-2-yl)benzimidazole and 2-amino-3,4-dihydro[1,3,5]triazino[1,2-a]benzimidazole hybrids were synthesized and evaluated for their in vitro antitumor activity at Nat...

In this paper green synthesis of an important commodity chemical lactamide has been undertaken using chitosan immobilized Rhodococcus pyridinivorans NIT-36 harbouring nitrile hydratase (NHase) enzyme. The cells immobilization (300 mg/g) is based on the partial entrapment of cells by suspension cross-linking technique facilitated by N, N′-Methylene bis-acrylamide. In the repeated-use experiments, the immobilized cells retained 80% of its initial activity when stored at 4 °C for 30 days. NHase activity of free and immobilized cells was studied over temperature ranging from 25 °C to 60 °C. The activity for free cells showed a sharp decline of 70% when the reaction temperature was elevated from 45 °C to 50 °C whereas chitosan immobilized cells retained their activity in the same temperature range. A fed-batch reaction was designed and the immobilized cells showed 100% similar enzymatic pattern for five consecutive rounds which gradually decreased in following cycles. A volumetric productivity of 20 g/L and catalytic productivity of 8.33 g/g dcw/h for lactamide were achieved.

Neoadjuvant treatment (NAT) can downstage breast cancer and can be utilized for different clinical applications. However, the response to NAT varies among individuals. Having effective biomarkers is important to optimize the treatment of breast cancer. Concentrations of biogenic amines have been found to show an association with cancer cell proliferation, but their clinical utility remains unclear. This study developed a postcolumn-infused internal standard (PCI-IS)-assisted liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method for profiling biogenic amines in human urine. Putrescine-d8 was selected as the PCI-IS to calibrate the errors caused by matrix effects in the urine sample. The optimized method was applied to investigate the association between changes in 14 amines and the therapeutic response to NAT in breast cancer patients. Urine samples were collected before initiation of chemotherapy (n = 60). Our results indicated that the levels of N1-acetylspermine, spermidine, norepinephrine, and dopamine were significantly higher in the responder group than the nonresponder group. These metabolites were incorporated with clinical factors to identify NAT responders, and the prediction model showed an area under the curve value of 0.949. These observations provide remarkable insights for future studies in elucidating the roles of biogenic amines in breast cancer. Additionally, the PCI-IS-assisted amine profiling method can facilitate these studies.

Background Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. Methods We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. Results Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. Conclusion Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

In a systematic study of light charged particle induced nuclear reactions we investigated the excitation functions of deuteron induced nuclear reactions on natural lutetium targets. Experimental excitation functions up to 50 MeV on high purity natLu were determined using the standard stacked foil activation technique. High resolution off-line gamma-ray spectrometry was applied to assess the activity of each foil. From the measured activity direct and/or cumulative elemental cross-section data for production of 171,172,173,175Hf, 171,172,173,174g,176m,177m,177gLu and 169Yb radioisotopes were determined. The experimental data were compared to results of the TALYS theoretical code taken from the TENDL databases and results of our calculations using the ALICE-IPPE-D and the EMPIRE-D codes. No earlier experimental data were found in the literature. Thick target yields for the investigated radionuclides were calculated from the measured excitation functions.

Background Discrepancies can occur with the use of clinical human immunodeficiency virus (HIV) diagnostic reagents for the HIV window period (WP; time from RNA to antibody detection by diagnostic or blood screening assays). Antiretroviral therapy (ART) during acute HIV infection can impact HIV-specific antibodies, antigens, and DNA/RNA detection. In this study, an HIV WP blood donor who initiated ART was monitored, evaluating the immunological and nucleic acid testing (NAT) results for early ART and discussing the potential effects on blood safety. Study design and methods This was a follow-up study of a HIV WP donor detected 36 hours after high-risk sexual behavior, who was subsequently treated with ART. Immunological and NAT methods were comparatively analyzed. Results The 4th generation HIV serologic assays were positive at Day 11, and the 3rd generation domestic anti-HIV assay was positive at Day 33. Individual donation (ID) NAT and minipool (MP) NAT of six samples were reactive, but 12-sample MP-NAT was nonreactive. ART resulted in a slow decline of HIV RNA, but HIV DNA was still detected on Day 757. Conclusion After ART, ID-NAT was more sensitive than MP-NAT or serologic detection; however, HIV DNA detection was more sensitive, with DNA but not RNA persistently detectable.

Background: Hepatitis B virus infection is one of the greatest threats to blood safety all over the world. The laboratory algorithm based on only the detection of hepatitis B surface antigen (HBsAg) leaves a gap for infected HBsAg negative donors to donate blood during the “window period” (WP) and late stages of infection. Objective: To estimate the frequency of the presence of HBV deoxyribonucleic acid (DNA) in HBsAg negative blood units screened using two different assays for HBsAg in a high endemic region. Methods: Frozen serum aliquot of 100 replacement blood donors who donated blood units that were HBsAg negative were retrieved and tested for HBV DNA. Sample positive for HBV DNA was sequenced by Sanger’s method, genotyped and the viral load was determined. Results: One sample (1%) was positive for HBV DNA. The HBV viral load of the sample was 768,000 IU/ml. The partial S-gene of the Hepatitis B virus isolated was genotype E using the NCBI viral genotyping tool. Conclusions: There is still a risk of HBV infected blood unit escaping detection when donor testing is limited to HBsAg screening. The use of NAT which can substantially reduce HBV infected blood donors from the donor pool should be considered. Keywords: Hepatitis B surface Antigen; Hepatitis B Virus; DNA; blood donors; blood safety.

MDR UPEC has become a global health challenge. Our study investigates the pairwise interactions among FOS, COL, NIT and TRI against 29 UPEC strains using the checkerboard method. The synergistic combinations are further evaluated for their bactericidal effects against the most resistant strain (MRS) using the time-kill method. The results showed that 100% of these strains were resistant to TRI and NIT, whereas 75.86% of them were susceptible to FOS and COL. Among all tested strains, only seven (strains were highly resistant to all used antibiotics. Remarkably, FOS/COL, COL/NIT and COL/TRI combinations represent the most effective synergistic (fractional inhibitory concentration index < 1) combinations against the seven strains at MICs lower than the susceptible breakpoint ranges, followed by FOS/NIT and FOS/TRI, which achieved synergistic interactions against 1/7 and 2/7 of these strains. Importantly, the bactericidal effects (reduction ≥ 3.0 Log10 cfu/ml) were only observed with FOS/COL, COL/NIT and COL/TRI combinations against MRS after 24 h of post-treatment. Our data suggested that FOS/COL, COL/NIT and COL/TRI combinations could be a promising option against MDR UPEC infections. Additionally, FOS/NIT and FOS/TRI probably represent a good option for MDR UPEC with lower MICs.

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.

Lactamide is used in the synthesis of lactic acid esters and as a solvent in many ecofriendly formulations. Till date lactamide synthesis was accomplished by employing chemical methods involving harsh reaction conditions while the enzyme-based approach ensures high purity lactamide synthesis under ambient environment without the intervention of acid byproducts using lactonitrile as substrate. Nitrile hydratase of Rhodococcus pyridinivorans NIT-36 was expressed by supplementing the growth medium with inducer i.e. lactamide. A 1.7-fold increase in enzyme activity was recorded by double feeding of the inducer. A further two-fold enhancement in enzymatic activity was achieved by the statistical optimization of process parameters for lactamide production by Response Surface Methodology. The substrate, lactonitrile inhibited the enzyme at higher concentrations, hence a fed-batch approach was adopted. The enzyme substrate ratio was standardized and the enzyme completely converted 15 mM of lactonitrile to lactamide in 30 minutes using 2 mg/ml resting cells. The fed-batch was performed at 1 L scale and 100% biotransformation of 360 mM of lactonitrile to lactamide was achieved at 45°C in 720 minutes. The catalytic productivity of 14.5 g/g dcw/h for lactamide formation was finally recorded in the 1 L fed-batch reaction.

Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452–459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism.

Here we have explored the effect of neoagarotetraose (NAT) on liver injury caused by intense exercise. Our results showed that NAT treatment obviously decreased liver weight (p < 0.01), improved the liver morphological structure, decreased ALT level (p < 0.05) and endotoxin (LPS) (p < 0.01). In addition, NAT could regulate bile acid profiles in feces and serum of mice, which indicated the potential of liver function, suggesting that NAT was effective to relieve intense exercise-induced liver injury. NAT could regulate the expression of colon genes. NAT tended to alter the microbial composition of mice under intense exercise. We uncovered the network interactions between liver traits and microbial communities in NAT treatment mice. Interestingly, our data indicated that intense exercise-induced liver injury may be related to Clostridiales. In summary, these results demonstrated that NAT relieved liver injury induced by intense exercise may be related to gut microbiota.

Arylamines constitute a large group of industrial chemicals detoxified by certain bacteria through conjugation reactions catalyzed by N-acetyltransferase (NAT) enzymes. NAT homologs, mostly from pathogenic bacteria, have been the subject of individual studies that do not lend themselves to direct comparisons. By implementing a practicable pipeline, we carried out a comparative investigation of 15 NAT homologs from 10 bacteria, mainly bacilli, streptomycetes, and one alphaproteobacterium. The new homologs were characterized for their sequence, phylogeny, predicted structural features, substrate specificity, thermal stability, and interaction with components of the enzymatic reaction. Bacillus NATs demonstrated the characteristics of xenobiotic metabolizing N-acetyltransferases, with the majority of homologs generating high activities. Nonpathogenic bacilli are thus proposed as suitable mediators of arylamine bioremediation. Of the Streptomyces homologs, the NAT2 isoenzyme of S. venezuelae efficiently transformed highly toxic arylamines, while the remaining homologs were inactive or generated low activities, suggesting that xenobiotic metabolism may not be their primary role. The functional divergence of Streptomyces NATs was consistent with their observed sequence, phylogenetic, and structural variability. These and previous findings support classification of microbial NATs into three groups. The first includes xenobiotic metabolizing enzymes with dual acetyl/propionyl coenzyme A (CoA) selectivity. Homologs of the second group are more rarely encountered, acting as malonyltransferases mediating specialized ecological interactions. Homologs of the third group effectively lack acyltransferase activity, and their study may represent an interesting research area. Comparative NAT enzyme screens from a broad microbial spectrum may guide rational selection of homologs likely to share similar biological functions, allowing their combined investigation and use in biotechnological applications. IMPORTANCE Arylamines are encountered as industrial chemicals or by-products of agrochemicals that may constitute highly toxic contaminants of soils and groundwaters. Although such chemicals may be recalcitrant to biotransformation, they can be enzymatically converted into less toxic forms by some bacteria. Therefore, exploitation of the arylamine detoxification capabilities of microorganisms is investigated as an effective approach for bioremediation. Among microbial biotransformations of arylamines, enzymatic conjugation reactions have been reported, including NAT-mediated N-acetylation. Comparative investigations of NAT enzymes across a range of microorganisms can be laborious and expensive, so here we present a streamlined methodology for implementing such work. We compared 15 NAT homologs from nonpathogenic, free-living bacteria of potential biotechnological utility, mainly Terrabacteria, which are known for their rich secondary and xenobiotic metabolism. The analysis allowed insights into the evolutionary and functional divergence of bacterial NAT homologs, combined with assessment of their fundamental structural and enzymatic differences and similarities.

The high demand for supplies during the COVID19-pandemic has generated several stock-out of material and essential reagents needed to meet the current high demand for diagnosis in the worldwide population. In this way, there is limited information regarding the performance of different virus transport medium (VTM) for nasopharyngeal swab sampling (NPS) aimed for SARS-CoV-2 detection. We compared the RT-qPCR amplification profile of four different commercial transport medium kits, including DNA/RNA Shield™, NAT™, VTM, and Phosphate-buffered saline (PBS) transport medium, for NPSs samples from Central Metropolitan Health Service, Santiago, Chile. The RT-qPCR showed a slight lower RNase P Cq value of the samples preserved and transported in DNA/RNA Shield™ compared to NAT medium. By contrast, a marked increase in the RNase P Cq value was registered in the samples transported with VTM compared to DNA/RNA Shield™ medium. For PBS-preserved NPS, the performance of two strategies were assessed due to the potential presence of any remaining active virus in the sample: (1) thermal inactivation; and (2) thermal inactivation treatment followed by RNA extraction. The heat inactivation showed a significantly lower Cq value for RNase P and viral ORF1ab Cq compared to the followed by RNA extraction. This study indicates that new medium alternatives could be used if supplies run out to diagnose COVID19.

Correction for ‘Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis’ by Rory F. Little et al., Nat. Prod. Rep., 2021, DOI: 10.1039/d1np00035g.

The disruption of Zn homeostasis has been linked with breast cancer development and progression. To enhance our understanding of changes in Zn homeostasis both inside and around the tumour microenvironment, Zn concentrations and isotopic compositions (δ66Zn) were determined in benign (BT) and malignant (MT) tumours, healthy tissue from reduction mammoplasty (HT), and histologically normal tissue adjacent to benign (NAT(BT)) and malignant tumours (NAT(MT)). Mean Zn concentrations in NAT(BT) are 5.5 µg g−1 greater than in NAT(MT) (p = 0.00056) and 5.1 µg g−1 greater than in HT (p = 0.0026). Zinc concentrations in MT are 12.9 µg g−1 greater than in HT (p = 0.00012) and 13.3 µg g−1 greater than in NAT(MT) (p

Natamycin (NAT) is the drug of choice for the treatment of fungal keratitis (FK). However, its inherent shortcomings, such as poor solubility, high dosing frequency, and long treatment cycle, need to be urgently addressed by designing a new delivery to widen its clinical utility. Growing research has confirmed that clotrimazole (CLZ) plays a significant role in fungal growth inhibition. Hence, coaxial electrospray (CO-ES) technology is used herein to prepare nano-systems with an average hydrodynamic particle size of 309-406 nm for the co-delivery of NAT and CLZ in chitosan (CTS) and poly(lactic-co-glycolic acid) (PLGA). The resulting NAT/CLZ@CTS/PLGA formulations were characterized by a transmission electron microscope (TEM) and in vitro release test. The results show that the formulations had obvious core-shell structures, uniform particle distribution, and also can sustain the release of drugs over 36 h. Furthermore, in vitro hemolysis, in vivo corneal irritation test, local allergenic test, and antifungal activity analyses are performed to evaluate the safety and efficiency of the formulations. Thus, good biosafety along with a significant anti-candidiasis effect are found in the NAT/CLZ@CTS/PLGA nanoparticles (NPs). Taken together, the results suggest that this design may provide a promising drug delivery system and a new option for the treatment of FK.

Nitrofurantoin (NIT) is a broad-spectrum bactericidal antibiotic used in the treatment of urinary tract infections. It is a pro-drug that once activated by nitroreductases goes on to inhibit bacterial DNA, RNA, cell wall, and protein synthesis. Previous work has suggested that NIT retains considerable activity against non-growing bacteria. Here we have found that Escherichia coli (E. coli) grown to stationary phase in minimal or artificial urine media are not susceptible to NIT. Supplementation with glucose under conditions where cells remained non-growing (other essential nutrients were absent) sensitized cultures to NIT. We conceptualized NIT sensitivity as a multi-input AND gate, and lack of susceptibility as an insufficiency in one or more of those inputs. The inputs considered were an activating enzyme, cytoplasmic abundance of NIT, and reducing equivalents required for NIT activation. We systematically assessed the contribution of each of these inputs and found that NIT import and the level of activating enzyme were not contributing factors to the lack of susceptibility. Rather, evidence suggested that the low abundance of reducing equivalents is why stationary-phase E. coli are not killed by NIT, and catabolites can re-sensitize those cells. We found that this phenomenon also occurred when using nitrofurazone, which established generality to the nitrofuran antibiotic class. In addition, we observed that NIT activity against stationary-phase uropathogenic E. coli (UPEC) could also be potentiated through metabolite supplementation. These findings suggest that the combination of nitrofurans with specific metabolites could improve the outcome of uncomplicated urinary tract infections (UTIs).

Vibrio vulnificus is a zoonotic bacterium that is capable of causing highly lethal diseases in humans; this pathogen is responsible for 95% of all seafood-related deaths in the United States. Arylamine N-acetyltransferases (NAT, E.C. 2.3.1.5) is a major family of xenobiotic-metabolizing enzymes that can biotransform aromatic amine chemicals. In this research, to evaluate the effect of NAT on acetyl group transformation in arylamine antibiotics, we first used sequence alignment to study the structure of V. vulnificus NAT [(VIBVN)NAT]. The nat gene encodes a protein of 260 amino acids, which has an approximate molecular mass of 30 kDa. Then we purified recombinant (VIBVN)NAT and determined the enzyme activity by PNPA and DTNB methods. The DTNB method indicates that this prokaryotic NAT has a particular substrate specificity towards aromatic substrates. However, (VIBVN)NAT lost most of its activity after treatment with high concentrations of urea and H2O2. In addition, we also explored the stability of the enzyme at different temperatures and pH values. In analyzing the influence of metal ions, the enzyme activity was significantly inhibited by Zn2+ and Cu2+. The kinetic parameters Km and Vmax were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the Tm, Tagg and size distribution of (VIBVN)NAT were observed. In particular, a molecular docking study on the structure of (VIBVN)NAT was conducted to understand its biochemical traits. These results showed that (VIBVN)NAT could acetylate various aromatic amine substrates and contribute to arylamine antibiotic resistance in V. vulnificus.

Glycans are responsible for many biological activities; however, their structures are incredibly diverse and complex often rendering the field of glycomics unsolvable by a single analytical technique. The development of multiple chemical derivatization strategies and bioinformatic software are responsible for some of the greatest analytical gains in the field of glycomics. The INLIGHT(™) strategy is a chemical derivatization technique using hydrazide chemistry to derivatize the reducing end of N-linked glycans and incorporates either a natural (NAT – (12)C(6)) or stable-isotope label (SIL – (13)C(6)) to carry out relative quantification. Here we present GlycoHunter, a user-friendly software created in MATLAB that enables researchers to accurately and efficiently process MS1 glycomics data where a NAT and SIL pair are generated for relative quantification, including but not limited to, INLIGHT(™). GlycoHunter accepts the commonly used data files formats imzML or mzXML, and effectively identifies all peak pairs associated with NAT and SIL labeled N-linked glycans using MS1 data. It also includes the ability to tailor the search parameters and export the results for further analysis using Skyline or Excel.

The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.

Major routes of metabolism for marketed drugs are predominately driven by enzyme families such as cytochromes P450 and UDP-glucuronosyltransferases. Less studied conjugative enzymes, like N-acetyltransferases (NATs), are commonly associated with detoxification pathways. However, in the clinic, the high occurrence of NAT polymorphism that leads to slow and fast acetylator phenotypes in patient populations has been linked to toxicity for a multitude of drugs. A key example of this is the observed clinical toxicity in patients who exhibit the slow acetylator phenotype and were treated with isoniazid. Toxicity in patients has led to detailed characterization of the two NAT isoforms and their polymorphic genotypes. Investigation in recombinant enzymes, genotyped hepatocytes, and in vivo transgenic models coupled with acetylator status-driven clinical studies have helped understand the role of NATs in drug development, clinical study design and outcomes, and potential roles in human disease models. The selected case studies herein document NAT enzyme kinetics to explore substrate overlap from two human isoforms, preclinical species considerations, and clinical genotype population concerns.

Here we highlight a sound and unique work reported by Chen and co-workers entitled “HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes” (Xiao et al., Nat. Chem. Biol. 2020, 16, 529). In this article, the authors identify, by means of a clever antibody-guided strategy, several small molecules as fusion inhibitors of HIV-1 replication acting at the membrane proximal external region (MPER) of the HIV-1 envelope (Env) spike. MPER, which was previously recognized as a vaccine target, emerges as a novel druggable target for the discovery of HIV-1 fusion inhibitors. The compounds (exemplified by dequalinium and dequalinium-inspired analogues) prevent the conformational changes of Env from the prefusion species to the intermediate states required for membrane fusion. This work not only paves the way to novel, specific and useful anti-HIV-1 inhibitors, but also discloses new therapeutic strategies against other infectious diseases., F.J.L. and M.-J.C. thank the Spanish MINECO/FEDER (SAF2015- 64629-C2-1-R, SAF2017-88107, MDM2017-0767, AEI-FEDER UE), MICIU (PID2019-104070RB-C21) and Generalitat de Catalunya (2017SGR1746) for financial support.

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z&rsquo, factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

Communications biology 4(1), 2 (1-12) (2021). doi:10.1038/s42003-020-01546-4, N-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development., Published by Springer Nature, London

International audience; Recent studies have revealed a possible link between the activities of polymorphic arylamine N-acetyltransferases (NATs) and energy metabolism. We used a Nat1/Nat2 double knockout (KO) mouse model to demonstrate that ablation of the two Nat genes is associated with modest, intermittent alterations in respiratory exchange rate. Pyruvate tolerance tests show that double KO mice have attenuated hepatic gluconeogenesis when maintained on a high-fat/high-sucrose diet. Absence of the two Nat genes also leads to an increase in the hepatic concentration of coenzyme A in mice fed a high-fat/high-sucrose diet. Our results suggest a modest involvement of NAT in energy metabolism in mice, which is consistent with the absence of major phenotypic deregulation of energy metabolism in slow human acetylators.

Severe corneal injury is one of the main causes of loss of visual function. Mesenchymal stem cells (MSCs) have the ability to repair damaged cells in vivo. The present study aimed to explore whether MSCs could function as a cell therapy tool to replace traditional methods to treat corneal injury. CD44 + /CD105 + mesenchymal stem cells isolated from mouse amniotic fluid (mAF-MSCs) were injected into mice after cryoinjury to induce corneal endothelial cell injury. Histopathological assays indicated that mAF-MSCs could promote the growth of corneal epithelial cells, reduce keratitis, and repair the corneal damage caused by low temperature. cDNA microarray analysis revealed that the mAF-MSCs affected the expression patterns of mRNAs related to cell proliferation and differentiation pathways in the mice after transplantation. The results of quantitative real-time PCR and western blotting revealed that NAT12, NAT10, and the ETV4/JUN/CCND2 signaling axis were elevated significantly in the mAF-MSC-transplantation group, compared with those in the phosphate-buffered saline-treated groups. High performance liquid chromatography–mass spectroscopy results revealed that mAF-MSCs could promote mRNA N4-acetylcytidine (ac4C) modification and high expression of N-acetyltransferase in the eyeballs. RNA immunoprecipitation-PCR results showed that a specific product comprising Vegfa, Klf4, Ccnd2, Jun, and Etv4 mRNA specific coding region sites could be amplified using PCR from complexes formed in mAF-MSC-transplanted samples cross-linked with anti-ac4C antibodies. Thus, mouse amniotic fluid MSCs could repair the mouse corneal cold injury by promoting the ETV4/JUN/CCND2 signal axis activation and improving its stability by stimulating N4-acetylcytidine modification of their mRNAs.

N-terminal acetylation (NTA) (see Glossary) is one of the most widespread protein modifications, which occurs on most eukaryotic proteins, but is significantly less common on bacterial and archaea proteins. This modification is carried out by a family of enzymes called N-terminal acetyltransferases (NATs) (see Glossary). To date, twelve NATs have been identified, harboring different composition, substrate specificity and, in some cases, modes of regulation. Recent structural and biochemical analysis of NAT proteins now allows for a comparison of their molecular mechanisms and modes of regulation, which are described here. Although sharing an evolutionarily conserved fold and related catalytic mechanism, each catalytic subunit employs unique elements to mediate substrate-specific activity and employ NAT-type specific auxiliary and regulatory subunits for their cellular functions.

EGCG and quercetin are flavonoids which usually co-exist in edible plants and they exhibit anti-diabetes effects. This study aimed to explore the mechanisms by which quercetin and EGCG synergistically protected pancreatic β-cells from streptozotocin-induced apoptosis. EGCG, quercetin, and their combinations (both 15 μM) all reversed STZ-induced cells damage and enhanced glucose-stimulated insulin secretion, with the combination being more effective than a single compound. At the molecular level, the EGCG-quercetin combination upregulated BCL-2 expression and caused a greater reduction in miR-16-5p level than EGCG alone or quercetin alone. Overexpression of miR-16-5p could offset the down-regulated apoptotic genes caused by the synergistic action of the combination. These findings suggest that EGCG and quercetin exert synergistic anti-diabetes effect, possibly via decreasing the expression of miR-16-5p that targets directly BCL-2. This is the first report on a miRNA-based mechanism underlying the synergistic protective effect of EGCG and quercetin against pancreatic cell damage.

To locate the site of a liver lesion by positron emission tomography (PET) imaging and then remove the lesion site under the guidance of fluorescence imaging, we designed an aggregate-induced emission (AIE)-based PET probe [nat/68Ga] 5 based on the effects of the reticuloendothelial system. By modifying derivatives of tetraphenylethene, we obtained a bifunctional chelating agent with AIE characteristics, which can complex with natGa3+ or 68Ga3+ at room temperature to form [natGa] 5 or [68Ga] 5. [natGa] 5 has typical AIE characteristics and can form nanomicelles when its concentration is greater than the critical micelle concentration (CMC). Cellular fluorescence confocal imaging experiments and radioactive uptake experiments showed that Kupffer cells had a high uptake of [nat/68Ga] 5 while HepG2 cells had lower uptake of [nat/68Ga] 5, indicating that liver function imaging was achieved by the phagocytosis of [nat/68Ga] 5 by Kupffer cells into the liver. A biodistribution study showed that [68Ga] 5 shows high accumulation in the liver after intravenous injection into rats. The in vivo PET imaging profile demonstrated an excellent liver to muscle ratio. Thus, [nat/68Ga] 5 could potentially be used as a liver function PET imaging agent for clinical diagnosis and intraoperative navigation.

An antifungal low-cost film for cheese packaging was developed using a tailor-made coating process by direct natamycin (Nat) spraying onto heat softened polyethylene film surface. Two spraying systems Nat/ethanol solution and Nat/n-heptane suspension with the same Nat concentration were explored. To select the adequate system, differences in coating efficiency regarding shape, size, dispersion and distribution of particles, as well as polyethylene film/Nat adhesion and antifungal activity of both modified films were assessed and compared. Besides, the suitability of final product related to its use as food flexible packaging was considered. The results indicate that Nat coating is achieved either by direct particle incorporation (from a suspension) or by its in-situ formation (from a solution). Both kind of Nat coated films have shown antifungal effectiveness against A. niger even after severe extraction treatments. The proposed versatile process is feasible to be industrially scaled and allows to obtain an antifungal film suitable for cheese packaging that can be intensively handled without losing their activity. Fil: Grafia, Ana Luisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentina Fil: Vazquez, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Bianchinotti, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Barbosa, Silvia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentina

The prevalence of hepatitis C virus (HCV) is highest in Egypt compared to other countries. Nucleic acid amplification test (NAT) allows detection of HCV early during the course of infection. Unfortunately, NAT is more expensive than ELISA, thus its routine use as a screening tool for blood products or in clinical practice is quite limited. The aim of this study was to compare two common RT-PCR methods, TaqMan probe technique and SYBR Green method in quantitative detection of HCV RNA for diagnosis and follow up of HCV patients. Among the recruited 220 HCV patients, 154 (70%) were HCV-RNA positive by both the techniques, while 24 (10.9%) were negative by both techniques. On the other hand, 40 (18.2%) cases were HCV RNA positive only by SYBR Green technique, and 2 (0.9%) only by TaqMan probe technique. Forty (20.4%) of the 196 chronic HCV cases were HCV-RNA positive by SYBR Green but negative by TaqMan probe technique.Conclusion: This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications.Keywords: HCV, PCR, SYBR Green 1, TaqMan probe, Viral load

Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (1) and -F (2) from the fermentation extract of Streptomyces conglobatus. The NAT biosynthetic gene cluster ( nat BGC) was identified by genome sequencing and bioinformatics analysis. nat BGC includes two nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) gene, and a gene cassette (10 genes), of which the encoded enzymes share high homology to the ones responsible for 3-formamidosalicylate (3-FAS) biosynthesis in the antimycin biosynthetic pathway. Heterologous expression of the partial nat BGC without the 3-FAS gene cassette in the antimycin producer, Streptomyces albus J1074, results in the production of 1 and 2, suggesting that the nat BGC indeed directs NATs biosynthesis. Targeted in-frame deletion of the reductase gene ( natE) abolished the production of 1 and 2 but accumulated two NAT derivatives, the known NAT-H (3) and a new NAT-I (4). Biochemical verification demonstrated that the recombinant NatE indeed catalyzes an NADPH-dependent reaction of 3 or 4 to 1 or 2, respectively. Compound 3 presented significantly stronger activities against eight cancer cell lines than the ones using cisplatin, the clinical chemotherapy medicine. In particular, 3 displayed 559- and 57-fold higher activity toward human melanoma and cervix epidermoid carcinoma cells, respectively, compared with cisplatin. The new derivative, 4, was 1.5- to 10.9-fold more active than cisplatin toward five cancer cell lines. The evaluation of NATs biosynthesis depicted here will pave the way to generate new NAT derivatives through rational pathway engineering.

64Cu is one of the most beneficial radionuclide that can be used as a theranostic agent in Positron Emission Tomography (PET) imaging. In this current work, 64Cu was produced with zinc oxide nanoparticles (natZnONPs) and zinc oxide powder (natZnO) via the 64Zn(n,p)64Cu reaction in Tehran Research Reactor (TRR) and the activity values were compared with each other. The theoretical activity of 64Cu also was calculated with MCNPX-2.6 and the cross sections of this reaction were calculated by using TALYS-1.8, EMPIRE-3.2.2 and ALICE/ASH nuclear codes and were compared with experimental values. Transmission Electronic Microscopy (TEM), Scanning Electronic Microscopy (SEM) and X-Ray Diffraction (XRD) analysis were used for samples characterizations. From these results, it's concluded that 64Cu activity value with nanoscale target was achieved more than the bulk state target and had a good adaptation with the MCNPX result.

Background Effectiveness of a flow-based treatment device using riboflavin photochemistry was demonstrated by cytopathic effect method using indicator viruses. However, inactivation efficacy against real blood-borne viruses needs to be evaluated, especially at nucleic acid level. Material and Methods Special plasma samples with varying concentrations of blood-borne virus were selected using a strict blood selection procedure and were treated with device treatment (DT). Nucleic acid test (NAT) using polymerase chain reaction fluorescence method was used to detect virus copies. Results The NAT value of 4325 in plasma with high Hepatitis B Virus (HBV) concentrations decreased to 1330 with DT. After 100-fold dilution, the NAT value was below the NAT detection limits with DT compared with 23.0 that without DT. The NAT value of 61.9 in plasma with medium HBV concentrations decreased to 37.8 with DT, and after 10-fold dilution, the NAT value was below the NAT detection limits with DT compared with below 20 that without DT. The Ct values of plasma with low concentrations of blood-borne viruses were below the NAT detection limits with DT. Conclusion There was a dose effect with DT which was effective in blood-borne viruses damaging nucleic acids to a level below the NAT detection limits.

Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA. Here, we describe and validate a new strategy to generate large-scale amounts of RBC-derived EVs for the delivery of RNA drugs, including antisense oligonucleotides, Cas9 mRNA, and guide RNAs. RNA drug delivery with RBCEVs shows highly robust microRNA inhibition and CRISPR–Cas9 genome editing in both human cells and xenograft mouse models, with no observable cytotoxicity.

This protocol is an extension to: Nat. Protoc. 10, 1643-1669 (2015); doi:10.1038/nprot.2015.103; published online 01 October 2015RNAs play key roles in many cellular processes. The underlying structure of RNA is an important determinant of how transcripts function, are processed, and interact with RNA-binding proteins and ligands. RNA structure analysis by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) takes advantage of the reactivity of small electrophilic chemical probes that react with the 2'-hydroxyl group to assess RNA structure at nucleotide resolution. When coupled with mutational profiling (MaP), in which modified nucleotides are detected as internal miscodings during reverse transcription and then read out by massively parallel sequencing, SHAPE yields quantitative per-nucleotide measurements of RNA structure. Here, we provide an extension to our previous in vitro SHAPE-MaP protocol with detailed guidance for undertaking and analyzing SHAPE-MaP probing experiments in live cells. The MaP strategy works for both abundant-transcriptome experiments and for cellular RNAs of low to moderate abundance, which are not well examined by whole-transcriptome methods. In-cell SHAPE-MaP, performed in roughly 3 d, can be applied in cell types ranging from bacteria to cultured mammalian cells and is compatible with a variety of structure-probing reagents. We detail several strategies by which in-cell SHAPE-MaP can inform new biological hypotheses and emphasize downstream analyses that reveal sequence or structure motifs important for RNA interactions in cells.