TEX15 associates with MILI and silences transposable elements in male germ cells.

DNA methylation is a major silencing mechanism of transposable elements (TEs). Here we report that TEX15, a testis-specific protein, is required for TE silencing. TEX15 is expressed in embryonic germ cells and functions during genome-wide epigenetic reprogramming. The Tex15 mutant exhibits DNA hypomethylation in TEs at a level similar to Mili and Dnmt3c but not Miwi2 mutants. TEX15 is associated with MILI in testis. As loss of Tex15 causes TE desilencing with intact piRNA production, our results identify TEX15 as a new essential epigenetic regulator that may function as a nuclear effector of MILI to silence TEs by DNA methylation.

In vivo PIWI slicing in mouse testes deviates from rules established in vitro.

Argonautes are small RNA-binding proteins, with some having small RNA-guided endonuclease (slicer) activity that cleaves target nucleic acids. One cardinal rule that is structurally defined is the inability of slicers to cleave target RNAs when nucleotide mismatches exist between the paired small RNA and the target at the cleavage site. Animal-specific PIWI clade Argonautes associate with PIWI-interacting RNAs (piRNAs) to silence transposable elements in the gonads, and this is essential for fertility. We previously demonstrated that purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that target sites with cleavage-site mismatches at the 10th and 11th piRNA nucleotides are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are base-paired perfectly with the piRNA. Additionally, we find that pachytene piRNA-guided slicing in both these situations failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis.

Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus.

Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.

Genetic Deficiency of Mtdh Gene in Mice Causes Male Infertility via Impaired Spermatogenesis and Alterations in the Expression of Small Non-coding RNAs.

Increased expression of metadherin (MTDH, also known as AEG-1 and 3D3/LYRIC) has been associated with drug resistance, metastasis, and angiogenesis in a variety of cancers. However, the specific mechanisms through which MTDH is involved in these processes remain unclear. To uncover these mechanisms, we generated Mtdh knock-out mice via a targeted disruption of exon 3. Homozygous Mtdh knock-out mice are viable, but males are infertile. The homozygous male mice present with massive loss of spermatozoa as a consequence of meiotic failure. Accumulation of γ-H2AX in spermatocytes of homozygous Mtdh knock-out mice confirms an increase in unrepaired DNA breaks. We also examined expression of the DNA repair protein Rad18, which is regulated by MTDH at the post-transcriptional level. In testes from Mtdh exon 3-deficient mice, Rad18 foci were increased in the lumina of the seminiferous tubules. The Piwi-interacting RNA (piRNA)-interacting protein Mili was expressed at high levels in testes from Mtdh knock-out mice. Accordingly, genome-wide small RNA deep sequencing demonstrated altered expression of piRNAs in the testes of Mtdh knock-out mice as compared with wild type mice. In addition, we observed significantly reduced expression of microRNAs (miRNAs) including miR-16 and miR-19b, which are known to be significantly reduced in the semen of infertile men. In sum, our observations indicate a crucial role for MTDH in male fertility and the DNA repair mechanisms required for normal spermatogenesis.

MILI, a PIWI family protein, inhibits melanoma cell migration through methylation of LINE1.

MILI, a member of the PIWI/AGO gene family, has been well documented to maintain genome integrity by transposon silencing in animal germ cells. It has been reported to be selectively expressed in precancerous stem cells (pCSCs), tumor cell lines and various malignancies. However, the underlying mechanism remains largely unclear. Here, we found that MILI is expressed in the melanoma cell line B16 but not in the highly metastatic mouse melanoma model B16BL6. Interestingly, the knockdown of MILI in B16 could activate MAGEA expression and increase the cell migration ability, whereas the overexpression of MILI in B16BL6 could inhibit MAGEA expression and decrease the cell migration ability. Further investigations showed that MILI can methylate LINE1, which is crucial for MAGEA expression and melanoma cell migration. Our results provide a novel function of MILI in melanoma metastasis and tumor progression.

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

Maxillofacial gunshot wounds.

The majority of maxillofacial gunshot wounds are caused by suicide attempts. Young men are affected most often. When the lower one-third of the face is involved, airway patency (1.6% of the cases) and hemorrhage control (1.9% of the cases) are the two most urgent complications to monitor and prevent. Spinal fractures are observed with 10% of maxillary injuries and in 20% of orbital injuries. Actions to treat the facial gunshot victim need to be performed, keeping in mind spine immobilization until radiographic imaging is complete and any required spinal stabilization accomplished. Patients should be transported to a trauma center equipped to deal with maxillofacial and neurosurgery because 40% require emergency surgery. The mortality rate of maxillofacial injuries shortly after arrival at a hospital varies from 2.8% to 11.0%. Complications such as hemiparesis or cranial nerve paralysis occur in 20% of survivors. This case has been reported on a victim of four gunshot injuries. One of the gunshots was to the left mandibular ramus and became lodged in the C4 vertebral bone.

TDRD1 phase separation drives intermitochondrial cement assembly to promote piRNA biogenesis and fertility.

The intermitochondrial cement (IMC) is a prominent germ granule that locates among clustered mitochondria in mammalian germ cells. Serving as a key platform for Piwi-interacting RNA (piRNA) biogenesis; however, how the IMC assembles among mitochondria remains elusive. Here, we identify that Tudor domain-containing 1 (TDRD1) triggers IMC assembly via phase separation. TDRD1 phase separation is driven by the cooperation of its tetramerized coiled-coil domain and dimethylarginine-binding Tudor domains but is independent of its intrinsically disordered region. TDRD1 is recruited to mitochondria by MILI and sequentially enhances mitochondrial clustering and triggers IMC assembly via phase separation to promote piRNA processing. TDRD1 phase separation deficiency in mice disrupts IMC assembly and piRNA biogenesis, leading to transposon de-repression and spermatogenic arrest. Moreover, TDRD1 phase separation is conserved in vertebrates but not in invertebrates. Collectively, our findings demonstrate a role of phase separation in germ granule formation and establish a link between membrane-bound organelles and membrane-less organelles.

Intensifying heterogeneous TiO2 photocatalysis for bromate reduction using the NETmix photoreactor.

This work focuses on the intensification of BrO3- (200 µg L-1) reduction by TiO2-assisted heterogeneous photocatalysis, using the NETmix mili-photoreactor illuminated by UVA light-emitting diodes (UVA-LEDs). The mili-photoreactor was assembled in two configurations: i) catalyst deposition on the channels and chambers of a back stainless steel slab (SSS) and ii) catalyst deposition on the front borosilicate glass slab (BGS), allowing the study of front-side (FSI) and back-side (BSI) illumination mechanisms, respectively. The BrO3- reduction rate in aqueous solution was assessed as a function of: i) pH; ii) dissolved oxygen (DO); iii) addition of formic acid (CH2O2) as a sacrificial agent (SA); iv) photocatalyst film thickness; v) illumination mechanism; vi) irradiation intensity; vii) temperature; and viii) water matrix. Higher BrO3- reduction rates were observed using the FSI mechanism and lower pH values. Nitrogen injection (to eliminate DO) did not significantly improve the reaction rate and the addition of CH2O2 had a negative effect at pH 6.5. Neither temperature nor irradiance increase showed a considerable improvement on the reduction rate. Moreover, TiO2 film remains stable for at least 13 consecutive reactions without significant catalyst leaching. The chemically pre-treated fresh water (FW) matrix negatively affected the reaction rate when compared with the synthetic water (SW), under the best operational conditions (SSS: pH = 5.5, 287 mg of TiO2, 25 °C, SA absence, [DO] = 232-263 µM). This was associated with the presence of both inorganic and organic matter at much higher concentrations than BrO3-. Notwithstanding, heterogeneous TiO2 photocatalysis, using the NETmix mili-photoreactor, was successfully applied to fresh water, achieving [BrO3-] < 10 µg L-1 (guideline value) after 2-hour reaction.

Intensification of heterogeneous TiO2 photocatalysis using the NETmix mili-photoreactor under microscale illumination for oxytetracycline oxidation.

This study focuses on the intensification of heterogeneous TiO2 photocatalysis for the removal of a contaminant of emerging concern (CEC), oxytetracycline (OTC), as a polishing step of urban wastewaters, using an innovative NETmix mili-photoreactor under UVA-LEDs illumination. The effect of catalyst coated surface per reactor volume and the illumination mechanism, back-side (BSI) or front-side (FSI) irradiation, on OTC oxidation were evaluated. For that, a thin film of photocatalyst was uniformly deposited on the front borosilicate slab (BS) (BSI mechanism; 333 m2catalyst m-3reactor) or on the network of channels and chambers imprinted in the back stainless-steel slab (SSS) (FSI mechanism; 989 m2catalyst m-3reactor) using a spray system. OTC removal was also assessed as a function of TiO2 film thickness immobilized on both slabs. The photocatalyst reactivity in combination with photoreactor was significantly enhanced (3.4 times) from 0.64 to 2.19 mmolOTC m-3illuminated reactor volume s-1, when considering the BSI and FSI mechanisms, respectively. In addition, the influence of UVA-LEDs intensity on OTC oxidation rate was investigated. UVA-LEDs plates were placed on the top of the NETmix borosilicate window. Moreover, the effect of water matrix was assessed using a secondary effluent from an urban wastewater treatment plant fortified with OTC. OTC oxidation rate was only inhibited in about 1.3 times in the presence of the real matrix, showing the ability of the NETmix to overcome matrix effects due to its unique characteristics. Catalyst film stability over four consecutive reaction cycles was evaluated using synthetic and real matrices fortified with OTC.

Removal of bromate from drinking water using a heterogeneous photocatalytic mili-reactor: impact of the reactor material and water matrix.

The main goal of this study was to evaluate the removal of bromate from drinking water using a heterogeneous photocatalytic mili-photoreactor, based on NETmix technology. The NETmix mili-reactor consists of a network of channels and chambers imprinted in a back slab made of acrylic (AS) or stainless steel (SSS) sealed, through mechanical compression and o-rings, with an UVA-transparent front borosilicate glass slab (BGS). A plate of UVA-LEDs was placed above the BGS window. TiO2-P25 thin films were immobilized on the BGS (back-side illumination, BSI) or SSS (front-side illumination, FSI) by using a spray deposition method. The photoreduction rate of a 200 µg L-1 (1.56 µM) BrO3- solution was assessed taking into account the following: (i) catalyst film thickness, (ii) catalyst coated surface and illumination mechanism (BSI or FSI), (iii) solution pH, (iv) type and dose of sacrificial agent (SA), (v) reactor material, and (vi) water matrix. In acidic conditions (pH 3.0) and in the absence of light/catalyst/SA, 28% and 36% of BrO3- was reduced into Br- only by contacting with AS and SSS during 2-h, respectively. This effect prevailed during BSI experiments, but not for FSI ones since back SSS was coated with the photocatalyst. The results obtained have demonstrated that (i) the molar rate of disappearance of bromates was similar to the molar rate of formation of bromides; (ii) higher BrO3- reduction efficiencies were reached in the presence of an SA using the FSI at pH 3.0; (iii) formic acid ([BrO3-]:[CH2O2] molar ratio of 1:3) presented higher performance than humic acids (HA = 1 mg C L-1) as SA; (iv) high amounts of HA impaired the BrO3- photoreduction reaction; (v) SSS coated catalyst surface revealed to be stable for at least 4 consecutive cycles, keeping its photonic efficiency. Under the best operating conditions (FSI, 18 mL of 2% wt. TiO2-P25 suspension, pH 3.0), the use of freshwater matrices led to (i) equal or higher reaction rates, when compared with a synthetic water in the absence of SA, and (ii) lower reaction rates, when compared with a synthetic water containing formic acid with a [BrO3-]:[CH2O2] molar ratio of 1:3. Notwithstanding, heterogeneous TiO2 photocatalysis, using the NETmix mili-reactor can be used to promote the reduction of BrO3- into Br-, attaining concentrations below 10 µg L-1 (guideline value) after 2-h reaction. Graphical Abstract .

Exonuclease Domain-Containing 1 Enhances MIWI2 piRNA Biogenesis via Its Interaction with TDRD12.

PIWI proteins and their associated small RNAs, called PIWI-interacting RNAs (piRNAs), restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most MILI piRNAs are derived via a slicer-independent pathway, MILI slicing loads MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1-/-;Tdrd12+/-), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, leading to infertility. Thus, EXD1 enhances MIWI2 piRNA biogenesis via a functional interaction with TDRD12.

Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function.

Small RNAs called PIWI-interacting RNAs (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by the PIWI protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing PIWI proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway.