Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response

BackgroundGrowth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood.MethodsThe effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation.ResultsThe functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection.ConclusionsThe growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model.General SignificanceThe increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.     

Preconcentration of organic solutes in urine by bubble bursting

Introduction

The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis.

Objectives

We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples.

Method

Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS).

Results

The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol.

Conclusion

Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders).

Graphical Abstract

Urine desalting and preconcentration in bursting bubbles.

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Trinucleotide’s quadruplet symmetries and natural symmetry law of DNA creation ensuing Chargaff’s second parity rule

For almost 50 years the conclusive explanation of Chargaff’s second parity rule (CSPR), the equality of frequencies of nucleotides A=T and C=G or the equality of direct and reverse complement trinucleotides in the same DNA strand, has not been determined yet. Here, we relate CSPR to the interstrand mirror symmetry in 20 symbolic quadruplets of trinucleotides (direct, reverse complement, complement, and reverse) mapped to double-stranded genome. The symmetries of Q-box corresponding to quadruplets can be obtained as a consequence of Watson–Crick base pairing and CSPR together. Alternatively, assuming Natural symmetry law for DNA creation that each trinucleotide in one strand of DNA must simultaneously appear also in the opposite strand automatically leads to Q-box direct-reverse mirror symmetry which in conjunction with Watson–Crick base pairing generates CSPR. We demonstrate quadruplet’s symmetries in chromosomes of wide range of organisms, from Escherichia coli to Neanderthal and human genomes, introducing novel quadruplet-frequency histograms and 3D-diagrams with combined interstrand frequencies. These “landscapes” are mutually similar in all mammals, including extinct Neanderthals, and somewhat different in most of older species. In human chromosomes 1–12, and X, Y the “landscapes” are almost identical and slightly different in the remaining smaller and telocentric chromosomes. Quadruplet frequencies could provide a new robust tool for characterization and classification of genomes and their evolutionary trajectories.     

Diethyl‐Ether Flower Washings of Dianthus cruentus Griseb. (Caryophyllaceae): Derivatization Reactions Leading to the Identification of New Wax Constituents

Some carnation species (Dianthus spp., Caryophyllaceae) exhibit a strong resistance to drought stress that is connected with the increased surface wax formation. Wax composition is unknown for the majority of Dianthus spp. Herein, mass spectral and gas chromatographic data, in combination with synthesis and chemical transformations (transesterification and synthesis of dimethyl disulfide adducts), enabled the identification of 151 constituents of diethyl‐ether washings of fresh flowers of Dianthus cruentus Griseb . from Serbia. The flower wax contained, along with the dominant ubiquitous long‐chain n‐alkanes, homologous series of n‐ and branched (iso‐ and anteiso‐) long‐chain hexyl alkanoates/alkenoates and alkyl/alkenyl benzoates. The branching position in the mentioned hexyl esters was probed by synthesis of esters of three isomeric hexanols that were spectrally characterized (¹H‐ and ¹³C‐NMR, IR, MS). The washings also contained long‐chain (Z)‐ and (E)‐alkenes (C₂₃?C₃₅) with several different double bond regiochemistries. Fifty‐five of these constituents (eight hexyl esters, two benzoates, and forty‐five alkenes) were detected for the first time in Plantae, while ten of these represent completely new compounds. The rare occurrence of these wax constituents makes them possible chemotaxonomic markers of this particular Dianthus sp.     

HPV18 Utilizes Two Alternative Branch Sites for E6~*I Splicing to Produce E7 Protein

Human papillomavirus 18(HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6 E7 pre-mRNA. The E6 ORF region in the bicistronic E6 E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6~*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences(BPS) upstream of its 30 splice site, with an identical heptamer AACUA■C, for E6~*I splicing. One heptamer has a branch site adenosine(underlined) at nt 384 and the other at nt 388. E6~*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6~*I splicing prefers the 30 ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6~*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment.     

CRISPR‐LbCas12a‐mediated modification of citrus

Recently, CRISPR‐Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR‐LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc‐facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBE_PthA4‐CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBE_PthA4‐CsLOBP. A total of seven 35S‐LbCas12a‐transformed Duncan plants were generated, and they were designated as #D₃₅s1 to #D₃₅s7, and ten Yao‐LbCas12a‐transformed Duncan plants were created and designated as #D_yao1 to #D_yao10. LbCas12a‐directed EBE_PthA4‐CsLOBP modifications were observed in three 35S‐LbCas12a‐transformed Duncan plants (#D₃₅s1, #D₃₅s4 and #D₃₅s7). However, no LbCas12a‐mediated indels were observed in the Yao‐LbCas12a‐transformed plants. Notably, transgenic line #D₃₅s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off‐targets were observed. Therefore, CRISPR‐LbCas12a can readily be used as a powerful tool for citrus genome editing.