Mycoplasma infection of cancer cells enhances anti-tumor effect of oxidized methylcytidines.

Oxidized methylcytidines 5-hydroxymethyl-2'deoxycytidine (5hmdC) and 5-formy-2'deoxycytidine (5fdC) are deaminated by cytidine deaminase (CDA) into genome-toxic variants of uridine, triggering DNA damage and cell death. These compounds are promising chemotherapeutic agents for cancer cells that are resistant to pyrimidine derivative drugs, such as decitabine and cytarabine, which are inactivated by CDA. In our study, we found that cancer cells infected with mycoplasma exhibited a markedly increased sensitivity to 5hmdC and 5fdC, which was independent of CDA expression of cancer cells. In vitro biochemical assay showed that the homologous CDA protein from mycoplasma was capable of deaminating 5hmdC and 5fdC into their uridine form. Moreover, mycoplasma infection increased the sensitivity of cancer cells to 5hmdC and 5fdC, whereas administration of Tetrahydrouridine (THU) attenuated this effect, suggesting that mycoplasma CDA confers a similar effect as human CDA. As mycoplasma infection occurs in many primary tumors, our findings suggest that intratumoral microbes could enhance the tumor-killing effect and expand the utility of oxidized methylcytidines in cancer treatment.

Exploring boar sperm sialylation during capacitation using boronic acid-functionalized beads.

Sialic acid (SA), which usually occupies the terminal position of oligosaccharide chains in mammalian spermatozoa, has important functions in fertilization. Compared with other methods, such as lectin probing, boronic acid could recognize and bind SA with a higher affinity and specificity at pH 6.9. In this study, two boronic acid carriers, 3-aminophenylboronic acid-labeled fluorescent latex (CML-APBA) and magnetic beads (CMM-APBA were applied to explore surface sialylation profile and sialoglycoproteins of the boar sperm. There are three binding sections of CML-APBA on the head of ejaculated sperm: acrosomal region, equatorial segment and the head posterior, which are the major regions undergoing sialylation. After capacitation in vitro, two major binding patterns of CML-APBA exists on sperm head. On some spermatozoa, sialylation exists on the equatorial segment and the posterior head, whilst on other spermatozoa, sialylation occurs on the acrosomal region and equatorial segment. Flow cytometry analysis suggested that the level of sialylation on boar sperm membrane decreases after capacitation. Furthermore, using CMM-APBA, we pulled down sialylated proteins from spermatozoa. Among them, two decapacitation factors associating on sperm surface, AWN and PSP-1, were identified. The levels of the two proteins reduced during capacitation, which might contribute to the decrease of sialylation on boar sperm surface.

Asymmetric Total Synthesis of Lancifodilactone G Acetate. 1. Diastereoselective Synthesis of CDEFGH Ring System.

The stereoselective construction of the CDEFGH ring system of lancifodilactone G is described. The key steps in this synthesis are (i) ring-closing metathesis for formation of the oxa-bridged eight-membered ring; (ii) an intramolecular Pauson-Khand reaction for construction of the sterically congested F ring; and (iii) sequential cross-metathesis, hydrogenation, and lactonization reactions for installation of the anomerically stabilized bis-spiro ketal fragment of lancifodilactone G.

STING activation in TET2-mutated hematopoietic stem/progenitor cells contributes to the increased self-renewal and neoplastic transformation.

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain unclear. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in dysregulated hematopoiesis in mouse models. DNA damage arising in Tet2-deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn promotes the enhanced self-renewal and development of CH. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant hematopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the development of CH associated with TET2 mutations is powered through chronic inflammation dependent on the activated cGAS-STING pathway and that STING may represent a potential target for intervention of relevant hematopoietic diseases.

Poly(4-ketovalerolactone) from Levulinic acid: Synthesis and Hydrolytic Degradation.

We report here the synthesis of poly(4-ketovalerolactone) (PKVL) via ring-opening transesterification polymerization (ROTEP) of the monomer 4-ketovalerolactone (KVL, two steps from levulinic acid). The polymerization of KVL proceeds to high equilibrium monomer conversion (up to 96% in the melt) to give the semicrystalline polyketoester PKVL with low dispersity. PKVL displays glass transition temperatures of 7 °C and two melting temperatures at 132 and 148 °C. This polyester can be chemically recycled through hydrolytic degradation. Under aqueous neutral or acidic conditions, the dominating pathway for polyester hydrolysis is through backbiting from the chain end. Under basic conditions, mid-chain cleavage, accelerated by the ketone carbonyl group in the backbone, promotes the hydrolysis of nearby backbone ester bonds. The final hydrolysis product is 5-hydroxylevulinic acid, the ring opened hydrolysis product of KVL. PKVL was also observed to degrade under the action of a Brønsted acid to a bis-spirocyclic dilactone natural product altaicadispirolactone, which is a dimer of KVL. This constitutes a rare example of a one-step synthesis of a secondary metabolite of non-trivial structure in which a polymer was the starting material and the sole source of matter. Analogous ROTEP of the isomeric 4-membered lactone 4-acetyl-ß-propiolactone (APL) was also explored, although this chemistry was not as well-behaved as the KVL to PKVL polymerization.

Sulfonamide-Trapping Reactions of Thermally Generated Benzynes.

Reactions of tethered, tertiary sulfonamides with thermally generated benzynes are reported. Typically, the N-S bonds in the substrates cleave, and saturated heterocycles [tetrahydroquinolines ( n = 2) and indolines ( n = 1)] are formed. The process is accompanied by either sulfonyl transfer or desulfonylation from a zwitterionic intermediate, with the favored pathway being largely dependent upon the size (5- vs 6-membered) of the N-containing ring in the zwitterion.

Isomerizations of Propargyl 3-Acylpropiolates via Reactive Allenes.

Thermal isomerizations of various propargyl 3-acylpropiolates are described. Many result in the formation of 3-acylbutenolides. These reactions appear to proceed through intermediate 2,3-dehydropyrans (strained cyclic allenes), which then isomerize in a previously unobserved fashion. Competitive processes that provide additional mechanistic insights are also described.

Intramolecular Capture of HDDA-Derived Benzynes: (i) 6- to 12-Membered Ring Formation, (ii) Internally (vis-à-vis Remotely) Tethered Traps, and (iii) Role of the Rate of Trapping by the Benzynophile.

Reported here are studies that have established novel features of the hexadehydro-Diels-Alder reaction of substrates containing tethered trapping moieties. Products having new structural motifs can be created. (i) Medium-sized fused rings can be produced by varying the length of the tether. (ii) The tether can emanate from an atom within the linker unit that joins the 1,3-diyne and diynophile. (iii) The importance of the rate of trapping by the benzynophile is established.

Influence of outer membrane vesicles of Proteus mirabilis isolated from boar semen on sperm function.

This study focuses on the effect of outer membrane vesicles (OMVs) in gram-negative bacteria on boar sperm function during in vitro storage. In the 40 ejaculates collected from Guangzhong Black boar, six gram-negative bacterial species were detected by 16S rDNA sequencing, of which Proteus mirabilis was the main contaminating bacterium. The OMVs of P. mirabilis were isolated by gradient ultracentrifugation. To reveal the effect of OMVs on boar sperm, different OMV concentrations were added to the Modena medium during sperm storage at 17 °C. Even after 3 days of storage, it was noted that low OMV dose (<5 µg/mL) in the extender did not significantly reduce sperm quality as compared with that in the control semen samples; however, sperm motility and sperm morphology were significantly altered in the extender owing to a high OMV dose (>10 µg/mL). The relative ROS level successively increased with OMV dose in sperm samples and storage time. Meanwhile, OMVs dramatically elevated the mitochondrial potential of sperm. OMVs could bind with the sperm membrane to further influence the capacity of sperm-oocyte binding; they also increased the expression of LC3 and caspase 3 and decreased that of anti-apoptosis-related protein, Bcl2, in sperm. It was concluded that OMVs of P. mirabilis influenced the function of boar sperm by inducing sperm membrane reconstruction as well as autophagy and apoptosis of sperm.

UCHL1 expression and localization on testicular development and spermatogenesis of Chinese giant salamanders.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), which is extensively expressed in vertebrates, is a deubiquitinating enzymes that inhibits the degradation of proteins by reversing ubiquitination modification. Herein, a 1087-bp sequence encoding UCHL1 was identified from the Chinese giant salamander (CGS; Andrias davidianus). The coding sequences (CDS) of UCHL1 encoded a putative poly peptide of 222 amino acids. The CGS UCHL1 isoforms were more related to their human and mouse counterparts. The phylogenic tree of vertebrate UCHL1 indicated that CGS UCHL1 has the closest relationship with human UCHL1 (up to 73.99 %). Before the gonads of male CGSs matured, the peak level of UCHL1 expression in testes appeared in 3-year-old CGSs according to RT-qPCR and western blot. In adult testes, the level of UCHL1 protein was lower in the breeding period than in the post-breeding period, whereas the level of UCHL1 protein in interstitial fluid of adult CGS testes was higher during the breeding period than during the post-breeding period. In testicular seminiferous lobules in the developmental stage of CGSs, immunohistochemistry displayed three kinds of localizing patterns of UCHL1, including nuclear localization at half year old, cytoplasmic localization from one year to three years old, and extracellular localization in adult. In testicular seminiferous lobules of adult CGS, the different developmental germ cells were separated by cysts containing UCHL1 protein, but UCHL1 did not localize on the mature sperm. The results showed that extracellular UCHL1 loaded on exosomes, as a component of the homogeneous germ cell cysts, could regulate the synchronous development of sperm in testes of adult CGS.

Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane.

Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity.

Autophagy and apoptosis induced by Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV).

The outbreak of Chinese Giant Salamander (Andrias davidianus, CGS) Iridovirus (CGSIV) caused massive death of CGSs. However, some CGSs with low level of CGSIV usually could survive. In our study, major capsid protein (MCP) DNA replicates of CGSIV in shedding skin were employed to assess the relative content of CGSIV in the living CGSs by qPCR. Furthermore, the examinations of autophagy and apoptosis in CGSs in vivo and in the primary renal cells in vitro were performed, respectively. The results showed that the relative contents of CGSIV in the shedding skin could reflect those in liver, spleen, and kidney of the CGSs. In these tissues of the CGSs with low-level replicates of CGSIV, there were not obviously macroscopic lesions. But the irregularly-shaped vesicles perhaps involving in autophagosome were observed by transmission electron microscopy (TEM). The LC3B protein displayed uneven distribution by Immunohistochemistry and the level mRNA of Atg5 was higher in these tissues than that in the tissues of healthy CGSs using qRT-PCR. Meanwhile, the apoptosis also appeared in these tissues by TUNEL staining and higher level mRNA of type I IFN were detected in these tissues using qRT-PCR. Further, both the expression level of LC3B II protein and Atg5 mRNA increased significantly at 2h after the virus infected the primary renal cells from the health CGSs in vitro. In addition, apoptosis and type I IFN mRNA began to increase significantly at 4h after the virus infected the renal cells. It was suggested that autophagy may be a pivotal role for survival of CGSIV in the CGSs during early infection and the rapid proliferation of CGSIV could be inhibited by innate immune response and apoptosis.