Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2′-O-methyl modification at their 3′ end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3′–5′ exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa

Zebrafish germline development in presence and absence of a functional PIWI pathway

Germcells are responsible for the transmission of all genetic information from one generation to the next and engage a genome defense pathway that is based upon RNA interference. This socalled PIWI or piRNA pathway protects the genome by targeting transposon transcripts and renders them inactive. PIWI proteins and their interacting small RNAs, the piRNAs, therefore represent an ancient defense system against intergenomic parasites. In zebrafish, where germcells are specified by the inheritance of germplasm, information about maternal transposon activity is passed on via the maternally provided PIWI protein Ziwi, a component of germ plasm. This maternally provided piRNA pool is thought to represent a first line of defense against transposon activity in early germ cells. Little is known about what happens in early germ cells of zebrafish in the time between their arrival at the genital ridge and the time when a functional gonad is established. We find that in this timeframe primordial germ cells (PGCs) are going through a transition from a maternal to a zygotic program. This change is accompanied by massive morphological changes and the induction of a germline specific transcriptional program. This not only leads to the expression of germline genes but also large intergenetic regions, start to get expressed. PiRNAs produced in the adult gonads map to these regions, suggesting that they might represent piRNA clusters, similar to what is described in mouse and drosophila. In the first few days, not only these clusters become transcribed but also the piRNA pathway becomes active leading to the amplification of maternal piRNAs. The second chapter of this thesis is looking at this early germ cell development. What happens in gonads where the PIWI pathway is compromised is subject to chapters 3 and 4. Vasa and Tdrd9 are two conserved proteins involved in the PIWI pathway in other organisms and we find that in zebrafish, absence of either of these two proteins leads to the loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility

Zebrafish germline development in presence and absence of a functional PIWI pathway

Germcells are responsible for the transmission of all genetic information from one generation to the next and engage a genome defense pathway that is based upon RNA interference. This socalled PIWI or piRNA pathway protects the genome by targeting transposon transcripts and renders them inactive. PIWI proteins and their interacting small RNAs, the piRNAs, therefore represent an ancient defense system against intergenomic parasites. In zebrafish, where germcells are specified by the inheritance of germplasm, information about maternal transposon activity is passed on via the maternally provided PIWI protein Ziwi, a component of germ plasm. This maternally provided piRNA pool is thought to represent a first line of defense against transposon activity in early germ cells. Little is known about what happens in early germ cells of zebrafish in the time between their arrival at the genital ridge and the time when a functional gonad is established. We find that in this timeframe primordial germ cells (PGCs) are going through a transition from a maternal to a zygotic program. This change is accompanied by massive morphological changes and the induction of a germline specific transcriptional program. This not only leads to the expression of germline genes but also large intergenetic regions, start to get expressed. PiRNAs produced in the adult gonads map to these regions, suggesting that they might represent piRNA clusters, similar to what is described in mouse and drosophila. In the first few days, not only these clusters become transcribed but also the piRNA pathway becomes active leading to the amplification of maternal piRNAs. The second chapter of this thesis is looking at this early germ cell development. What happens in gonads where the PIWI pathway is compromised is subject to chapters 3 and 4. Vasa and Tdrd9 are two conserved proteins involved in the PIWI pathway in other organisms and we find that in zebrafish, absence of either of these two proteins leads to the loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility

Bone Graft Packing and Its Association with Bone Regeneration in Maxillary Sinus Floor Augmentations: Histomorphometric Analysis of Human Biopsies

Research in maxillary sinus floor augmentation (MSFA) focussed on the optimisation of microstructural parameters such as microporosity and particle size of bone substitute particles (BS). However, little is known about the impact of BS packing and the corresponding (void) interparticular space on bone regeneration. The aim of this study was to characterise the spatial distribution of BS and its association with BS integration 6 ± 1 months after MSFA. Histological thin-ground sections of 70 human sinus biopsies were histomorphometrically analysed: In serial zones of 100 µm proceeding from the sinus floor (SF) up to the apical end of the biopsy, we measured the distribution of BS particles within these zones in terms of volume (BSV/TV), number and size of BS particles, interparticle spacing (BS.Sp) and bone-to-BS contact. BS particles were not homogeneously distributed over the length of biopsies: The first 200 µm directly adjacent to the SF represented a zone poor in BS particles but with high osteogenic potential. Graft packing density increased from the SF towards the apical part of the AA. Integration of BS particles was inversely associated with the distance to the SF and the graft packing density. A high packing density through excessive compaction of BS particles should be avoided to optimise the macrostructural environment for bone regeneration

Reply to Schausberger, P. Not Seeing the Mites for the Hairs. Comment on “Möth et al. Unexpected Effects of Local Management and Landscape Composition on Predatory Mites and Their Food Resources in Vineyards. Insects 2021, 12, 180”

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Unexpected Effects of Local Management and Landscape Composition on Predatory Mites and Their Food Resources in Vineyards

Viticultural practices and landscape composition are the main drivers influencing biological pest control in vineyards. Predatory mites, mainly phytoseiid (Phytoseiidae) and tydeoid mites (Tydeidae), are important to control phytophagous mites (Tetranychidae and Eriophyidae) on vines. In the absence of arthropod prey, pollen is an important food source for predatory mites. In 32 paired vineyards located in Burgenland/Austria, we examined the effect of landscape composition, management type (organic/integrated), pesticide use, and cover crop diversity of the inter-row on the densities of phytoseiid, tydeoid, and phytophagous mites. In addition, we sampled pollen on vine leaves. Typhlodromus pyri Scheuten was the main phytoseiid mite species and Tydeus goetzi Schruft the main tydeoid species. Interestingly, the area-related acute pesticide toxicity loading was higher in organic than in integrated vineyards. The densities of phytoseiid and tydeoid mites was higher in integrated vineyards and in vineyards with spontaneous vegetation. Their population also profited from an increased viticultural area at the landscape scale. Eriophyoid mite densities were extremely low across all vineyards and spider mites were absent. Biological pest control of phytophagous mites benefits from less intensive pesticide use and spontaneous vegetation cover in vineyard inter-rows, which should be considered in agri-environmental schemes