Chemical hazard of robotic hull in-water cleaning discharge on coastal embryonic fish.

In-water cleaning (IWC) involves the removal of biofilms and foulants from the hull of a ship using brush or water jet. During IWC, several factors associated with the harmful chemical contaminants release to the marine environment, which can create "hotspots" of chemical contamination in coastal areas. To elucidate the potential toxic effects of IWC discharge, we investigated developmental toxicity in embryonic flounder, which are sensitive life stage to chemical exposure. Zinc and copper were the dominant metals, while zinc pyrithione was the most abundant biocide associated with IWC discharge in two remotely operated IWC. Discharge from IWC carried by both remotely operated vehicles (ROVs) produced developmental malformations including pericardial edema, spinal curvature, and tail-fin defects. In an analyses of differential gene expression profiles (fold-change of genes with a cutoff < 0.05) as assessed by high-throughput RNA sequencing, genes associated with muscle development were commonly and significantly changed. The gene ontology (GO) of embryos exposed to IWC discharge from ROV A activities highly enriched muscle and heart development, while cell signaling and transport were evident in embryos exposed to IWC discharge of ROV B. We analyzed the gene network by significant GO terms. In the network, TTN, MYOM1, CASP3, and CDH2 genes appeared to be key regulators of the toxic effects on muscle development. In embryos exposed to ROV B discharge, HSPG2, VEGFA, and TNF genes related to the nervous system pathway were affected. These results shed light on the potential impacts of muscle and nervous system development in non-target coastal organisms exposed to contaminants found in IWC discharge.

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

Effectiveness of nasotracheal intubation in anterior cervical surgery including C3 lesions.

OBJECTIVE: Anterior approach cervical surgery is widely used for accessing C3 lesions. When operating with an anterior approach, the surgical field is obstructed by mandible. Neck extension is popular method to secure better surgical field but risk devastating neurological damage. To overcome this limited surgical field without neck extension, we adopted nasotracheal intubation and evaluated its efficiency. METHODS: We retrospectively analyzed 16 patients who underwent anterior cervical discectomy or corpectomy of C3 lesions via nasotracheal intubation. We enrolled an additional 29 patients who underwent anterior cervical discectomy or corpectomy of C3 lesions via orotracheal intubation as a control group. All patients had been diagnosed with cervical spondylotic myelopathy or ossification of the posterior longitudinal ligament. We measured the mandibular-cervical angle, which is the angle between the lower mandibular line and anterior vertebral line. RESULTS: The mandibular-cervical angle was increased by 7.3 with nasotracheal intubation compared to orotracheal intubation. CONCLUSIONS: Nasotracheal intubation is an effective surgical option for securing the surgical field without neck extension in anterior cervical surgery including C3 lesions.

Overlapping and unique toxic effects of three alternative antifouling biocides (Diuron, Irgarol 1051®, Sea-Nine 211®) on non-target marine fish.

The use of alternative biocides has increased due to their economic and ecological relevance. Although data regarding the toxicity of commercial alternative biocides in marine organisms are accumulating, little is known about their toxic pathways or mechanisms. To compare the toxic effects of commercial alternative biocides on non-target pelagic fish (flounder) embryos, we investigated the adverse effects of developmental malformation and transcriptional changes. Three biocides including Diuron, Irgarol 1051® and Sea-Nine 211® produced a largely overlapping suite of developmental malformations, including tail-fin fold defects and dorsal body axis curvature. In our test, the potencies of these biocides were ranked in the following order with respect to malformation and mortalities: Sea-Nine 211®â€¯> Irgarol 1051®â€¯> Diuron. Consistent with the toxicity rankings, the expression of genes related to heart formation was greater in embryonic flounder exposed to Sea-Nine 211® than in those exposed to Irgarol 1051® or Diuron, while expression of genes related to fin malformation was greater in the Irgarol 1051® exposure group. In analyses of differential gene expression (DEG) profiles (fold change of genes with a cutoff P < 0.05) by high-throughput sequencing (RNA-seq), genes associated with nervous system development, transmembrane transport activity, and muscle cell development were significantly changed commonly. Embryos exposed to Diuron showed changes related to cellular protein localization, whereas genes associated with immune system processes were up-regulated significantly in embryos exposed to Irgarol 1051®. Genes related to actin filament organization and embryonic morphogenesis were up-regulated in embryos exposed to Sea-Nine 211®. Overall, our study provides a better understanding of the overlapping and unique developmental toxic effects of three commercial booster biocides through transcriptomic analyses in a non-target species, embryonic flounder.

Quercetin inhibits the poly(dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed human keratinocytes.

Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1ß and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10 µM of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10 µM, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes.

West meets East: How do rainforest beetles become circum-Pacific? Evolutionary origin of Callipogon relictus and allied species (Cerambycidae: Prioninae) in the New and Old Worlds.

The longhorn beetle genus Callipogon Audinet-Serville represents a small group of large wood-boring beetles whose distribution pattern exhibits a unique trans-Pacific disjunction between the East Asian temperate rainforest and the tropical rainforest of the Neotropics. To understand the biogeographic history underlying this circum-Pacific disjunct distribution, we reconstructed a molecular phylogeny of the subfamily Prioninae with extensive sampling of Callipogon using multilocus sequence data of 99 prionine and four parandrine samples (ingroups), together with two distant outgroup species. Our sampling of Callipogon includes 18 of the 24 currently accepted species, with complete representation of all species in our focal subgenera. Our phylogenetic analyses confirmed the purported affinity between the Palearctic Callipogon relictus and its Neotropical congeners. Furthermore, based on molecular dating under the fossilized birth-death (FBD) model with comprehensive fossil records and probabilistic ancestral range reconstructions, we estimated the crown group Callipogon to have originated in the Paleocene circa 60 million years ago (Ma) across the Neotropics and Eastern Palearctics. The divergence between the Palearctic C. relictus and its Neotropical congeners is explained as the result of a vicariance event following the demise of boreotropical forest across Beringia at the Eocene-Oligocene boundary. As C. relictus represents the unique relictual species that evidentiates the lineage's expansive ancient distribution, we evaluated its conservation importance through species distribution modelling. Though we estimated a range expansion for C. relictus by 2050, we emphasize a careful implementation of conservation programs towards the protection of primary forest across its current habitats, as the species remains highly vulnerable to habitat disturbance.

Trace element analysis of three tissues from Eurasian otters (Lutra lutra) in South Korea.

Eurasian otters (Lutra lutra) are endangered worldwide, but the specific cause of their decline has not been determined. This study analyzed the concentrations of potentially toxic trace elements, including As, Cd, Pb, Hg, Se, Cu, Mn, and Zn, in the liver, kidney, and lung tissues of Eurasian otters in South Korea. There were high individual variations in the tissue concentrations of all the elements analyzed. The kidneys had the highest concentrations of Cd and Se among the three tissue groups, and the livers had the highest concentrations of Cu, Mn, Zn, and Hg. The Pb and As concentrations in the livers were not significantly different from those in the kidneys, and the lungs had the lowest concentrations of all the elements analyzed. The age-related bioaccumulation of Cd and Hg was evident in the three tissue groups, and of Se in the kidneys. The Pb concentration was higher in the livers of juveniles compared with those of adults and the Zn concentration was higher in the lungs of juveniles. There were no apparent gender differences in the concentrations of the elements analyzed among the tissue groups. The Se concentration correlated with the Hg concentration in the livers and kidneys, and with the Cd concentration in the kidneys. The Hg and Cd levels correlated in the three tissue groups. The Cu and Zn levels also correlated in the livers and kidneys. In general, the element concentrations were within the ranges reported by previous studies of this species from European countries, except for Cd and Hg, the levels of which were mostly lower than those reported previously. These findings may provide baseline information to facilitate the conservation of the Eurasian otter. To the best of our knowledge, this is the first available study of trace element concentrations in the tissues of Eurasian otters from South Korea or Asian countries.

Potential Threat of Microplastics to Zooplanktivores in the Surface Waters of the Southern Sea of Korea.

The potential impact of microplastic to zooplanktivores was assessed by measuring a ratio of neustonic microplastics to zooplankton by abundance in the southern sea of Korea. Neustonic microplastics and zooplankton (0.33-2 mm) were collected using a 330-µm mesh Manta trawl in Geoje eastern Bay and Jinhae Bay before and after the rainy season in 2012 and 2013. The mean microplastic to zooplankton ratios were 0.086 (May) and 0.022 (July) in 2012, and 0.016 (June) and 0.004 (July) in 2013, indicating that zooplanktivores could be more likely to feed on microplastics than natural preys before the rainy season in 2012 and 2013. In particular, the relatively high ratio occurred in a semi-enclosed bay characterized by a shipyard and a beach resort in Geoje Bay, and at stations close to a wastewater treatment plant and an aquaculture facility in Jinhae Bay before the rainy season. Among dominant microplastics and zooplankton before the rainy season, meroplankton of macrobenthos could be confused with paint particles in Geoje Bay, 2012, whereas Styrofoam could be mistaken as immature copepods by predators in Jinhae Bay, 2013. These observations suggest that zooplanktivores could be more likely to feed on microplastics than natural preys around Geoje and Jinhae Bays before the rainy season.

Large accumulation of micro-sized synthetic polymer particles in the sea surface microlayer.

Determining the exact abundance of microplastics on the sea surface can be susceptible to the sampling method used. The sea surface microlayer (SML) can accumulate light plastic particles, but this has not yet been sampled. The abundance of microplastics in the SML was evaluated off the southern coast of Korea. The SML sampling method was then compared to bulk surface water filtering, a hand net (50 µm mesh), and a Manta trawl net (330 µm mesh). The mean abundances were in the order of SML water > hand net > bulk water > Manta trawl net. Fourier transform infrared spectroscopy (FTIR) identified that alkyds and poly(acrylate/styrene) accounted for 81 and 11%, respectively, of the total polymer content of the SML samples. These polymers originated from paints and the fiber-reinforced plastic (FRP) matrix used on ships. Synthetic polymers from ship coatings should be considered to be a source of microplastics. Selecting a suitable sampling method is crucial for evaluating microplastic pollution.

Is it true that treatment in patients with Subaxial Cervical Spine Injury Classification System (SLICS) 4 is the surgeon's choice?

BACKGROUND: The Subaxial Cervical Spine Injury Classification System (SLICS) is a commonly used algorithm for diagnosing and managing subaxial cervical spine trauma. A SLIC score 4 suggests either surgery or non-surgically treatment depending on the surgeon's experience and patient's conditions. OBJECTIVE: Prognosis and treatment results were analyzed in patients with SLIC score 4. METHODS: The patients with SLIC score 4 were retrospectively reviewed from 2012 to 2019. Forty-one patients were included and divided into two groups: non-surgically treated and surgically treated. Demographic data and radiographs were analyzed. Statistical analysis was performed to determine the difference between the two clinical groups. RESULTS: Twenty-two patients were non-surgically treated, and nineteen patients were surgically treated. There was no neurological deterioration in both groups. However, there was no statistically significant difference in the last follow-up AISA and Nurick grade (p> 0.05). There was no significant difference in the number of patients who showed improvement when comparing the initial and the last follow-up neurological status (p> 0.05). CONCLUSION: Regardless of the treatment method, the spinal cord injury patients with SLICS point 4 showed a relatively good prognosis. Patients with SLIC score 4 could be treated non-surgically or surgically based on the surgeon's experience and factors associated with the patient's acute health status and chronic comorbidities.

Hidden underlying mechanisms for changes in mesozooplankton communities: Transport and eddy driven changes.

Mesozooplankton communities have been used extensively as reliable climate change indicators, mainly because of their rapid growth and sensitivity to environmental changes. This study explored the modifications in the taxonomic composition of the mesozooplankton community and the associated physical changes of transport-driven, eddy-driven, and marine heatwaves in the summers of the last 14 years (2009-2022) within the mixed layer of the Ulleung Basin in the East Sea/Japan Sea, where surface waters have rapidly warmed in recent decades. A slight increase was observed in the abundance of mesozooplankton from 2009 (3709 inds.m-3) to 2022 (4231 inds.m-3), with two notable peaks in 2015 (11,377 inds.m-3) and 2020 (11,184 inds.m-3), which was mainly attributed to the prevalence of Noctiluca scintillans. The first peak in 2015 showed thaliaceans to be the next dominant taxa, in which the southward direction of meandering in East Korea Warm Current (EKWC), presence of the Ulleung warm eddy, lower volume of the Western Channel (V-west) of the Korea Strait, and marine heatwaves (MHWs) did not occur. In contrast to the first peak, the second peak in 2020 showed Pyrocystis pseudonoctiluca to be the next dominant species, which may have been transported and advected by the strong V-west and eastward direction of the EKWC and the occurrence of MHWs that allowed the persistence of the subtropical species P. pseudonoctiluca. Overall, the significant increases in the second dominant mesozooplankton taxa appeared to be affected by physical changes, including transport or eddy-driven changes, along with the occurrence of strong V-west, the direction of the EKWC, and the occurrence of MHWs, which may synergistically influence the increase in the second dominant taxa during summer. This study highlights the complex interplay between notable variations in mesozooplankton communities and environmental factors, highlighting the potential consequences of different physical changes (transport-driven and eddy-driven) in this regional ocean.

Direct Measurement of the Thermal Expansion Coefficient of Epitaxial WSe2 by Four-Dimensional Scanning Transmission Electron Microscopy.

Current reports of thermal expansion coefficients (TEC) of two-dimensional (2D) materials show large discrepancies that span orders of magnitude. Determining the TEC of any 2D material remains difficult due to approaches involving indirect measurement of samples that are atomically thin and optically transparent. We demonstrate a methodology to address this discrepancy and directly measure TEC of nominally monolayer epitaxial WSe2 using four-dimensional scanning transmission electron microscopy (4D-STEM). Experimentally, WSe2 from metal-organic chemical vapor deposition (MOCVD) was heated through a temperature range of 18-564 °C using a barrel-style heating sample holder to observe temperature-induced structural changes without additional alterations or destruction of the sample. By combining 4D-STEM measurements with quantitative structural analysis, the thermal expansion coefficient of nominally monolayer polycrystalline epitaxial 2D WSe2 was determined to be (3.5 ± 0.9) × 10-6 K-1 and (5.7 ± 2) × 10-5 K-1 for the in- and out-of-plane TEC, respectively, and (3.6 ± 0.2) × 10-5 K-1 for the unit cell volume TEC, in good agreement with historically determined values for bulk crystals.

Characterization of Escherichia coli Strains for Novel Production of Plasmodium ovale Lactate Dehydrogenase.

Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, Plasmodium ovale morphologically resembles Plasmodium vivax, resulting in misidentification and confusion in diagnosis, and is responsible for malarial disease relapse due to the formation of hypnozoites. P. ovale receives relatively less attention compared to other major parasites, such as P. falciparum and P. vivax, primarily due to its lower pathogenicity, mortality rates, and prevalence rates. To efficiently produce lactate dehydrogenase (LDH), a major target for diagnosing malaria, this study used three Escherichia coli strains, BL21(DE3), BL21(DE3)pLysS, and Rosetta(DE3), commonly used for recombinant protein production. These strains were characterized to select the optimal strain for P. ovale LDH (PoLDH) production. Gene cloning for recombinant PoLDH production and transformation of the three strains for protein expression were performed. The optimal PoLDH overexpression and washing buffer conditions in nickel-based affinity chromatography were established to ensure high-purity PoLDH. The yields of PoLDH expressed by the three strains were as follows: BL21(DE3), 7.6 mg/L; BL21(DE3)pLysS, 7.4 mg/L; and Rosetta(DE3), 9.5 mg/L. These findings are expected to be highly useful for PoLDH-specific diagnosis and development of antimalarial therapeutics.

Size-dependent effects of micro polystyrene particles in the marine copepod Tigriopus japonicus.

We investigated the effects of three sizes of polystyrene (PS) microbeads (0.05, 0.5, and 6-µm diameter) on the survival, development, and fecundity of the copepod Tigriopus japonicus using acute and chronic toxicity tests. T. japonicus ingested and egested all three sizes of PS beads used and exhibited no selective feeding when phytoplankton were added. The copepods (nauplius and adult females) survived all sizes of PS beads and the various concentrations tested in the acute toxicity test for 96 h. In the two-generation chronic toxicity test, 0.05-µm PS beads at a concentration greater than 12.5 µg/mL caused the mortality of nauplii and copepodites in the F0 generation and even triggered mortality at a concentration of 1.25 µg/mL in the next generation. In the 0.5-µm PS bead treatment, despite there being no significant effect on the F0 generation, the highest concentration (25 µg/mL) induced a significant decrease in survival compared with the control population in the F1 generation. The 6-µm PS beads did not affect the survival of T. japonicus over two generations. The 0.5- and 6-µm PS beads caused a significant decrease in fecundity at all concentrations. These results suggest that microplastics such as micro- or nanosized PS beads may have negative impacts on marine copepods.

Functional Anatomy of the Spinal Tracts Based on Evolutionary Perspectives.

The development of spinal cord represents evolutionary progression. The primitive tract is responsible for functions related to basic survival such as locomotion. In contrast, the developed tracts are involved in perceiving the external environment and controlling conscious movements. There are also differences in the arrangement of spinal tracts between the 2 categories. Tracts serving developed functions are located in the deep layer of the lateral funiculus, whereas primitive tracts occupy other areas. Decussation correlates with tract pathways, with primitive tracts projecting ipsilaterally and developing tracts decussating early. Understanding these principles provides insights into spinal tract organization.

Efficacy of a newly designed helical-shaped 3D-printed titanium cage for cervical vertebral defect healing in rabbits.

Three-dimensional (3D) printed titanium (Ti-6Al-4V alloy) cages are widely used for spinal fusion applications. However, the structural design and shape of the cages are a major determinant of the optimal clinical outcome. In this study, we constructed a newly designed 3D-printed helical-shaped titanium cage (HTC) with a flexible body, and compared its healing and fusion efficacy in cervical vertebral defects after corpectomy in rabbits to that of a 3D-printed traditional titanium cage (TTC). We performed radiological examinations 1 and 16 weeks after TTC and HTC implantation. We assessed bone ingrowth in TTC and HTC using micro-computed tomography (micro-CT) and histological staining of tissue sections at 16 weeks. The radiographic data showed that the HTC-implanted group had better restoration of vertebral height than the TTC group, indicating a lower risk of cage subsidence. The micro-CT and histological observations showed that HTC promoted bone regeneration and osseointegration more effectively than TTC. Histomorphometry further revealed significant new bone formation in the HTC group compared to the TTC group. These findings demonstrate that HTC has better healing and bone fusion effects than TTC in cervical vertebral defects in rabbits, indicating its potential clinical value.

Effects on Bioaccumulation, Growth Performance, Hematological Parameters, Plasma Components, and Antioxidant Responses in Starry Flounder (Platichthys stellatus) Exposed to Dietary Cadmium and Ascorbic Acid.

This study evaluates the toxic effects of dietary Cd and mitigative effects of AsA supplementation by measuring the growth performance, bioaccumulation, hematological parameters, plasma components, and antioxidant responses of Starry flounder (Platichthys stellatus). Platichthys stellatus (mean weight, 69.5 ± 1.4 g; mean length, 18.2 ± 0.21 cm) was fed with dietary cadmium-ascorbic acid (Cd-AsA) composed of C0A0, C0A500, C0A1000, C40A0, C40A500, C40A1000, C80A0, C80A500, and C80A1000 mg of Cd-AsA per kg diet for four weeks. Our results showed that Cd accumulation significantly increased in proportion to the Cd concentration, where the highest levels were observed in the intestine, followed by the kidney, liver, and gills. Dietary AsA significantly mitigated the Cd accumulation in all tissues, and the reduction in Cd accumulation was proportional to the increase in AsA concentration. Dietary Cd has adverse effects on growth performance (body weight gain, specific growth rate, feed conversion ratio, and hepatosomatic index) and can alter the hematological parameters (red blood cell count, hematocrit, and hemoglobin), plasma components (glucose, total protein, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase), and antioxidant responses (superoxide dismutase, catalase, glutathione S-transferase, and glutathione). Dietary AsA restored the decreased growth performance parameters and the altered hematological parameters, plasma components, and antioxidant responses caused by the dietary Cd exposure. The results of this study showed that dietary Cd is toxic to P. stellatus, while dietary AsA is effective in mitigating the toxic effects of Cd.

Heterosynaptic MoS2 Memtransistors Emulating Biological Neuromodulation for Energy-Efficient Neuromorphic Electronics.

Heterosynaptic neuromodulation is a key enabler for energy-efficient and high-level biological neural processing. However, such manifold synaptic modulation cannot be emulated using conventional memristors and synaptic transistors. Thus, reported herein is a three-terminal heterosynaptic memtransistor using an intentional-defect-generated molybdenum disulfide channel. Particularly, the defect-mediated space-charge-limited conduction in the ultrathin channel results in memristive switching characteristics between the source and drain terminals, which are further modulated using a gate terminal according to the gate-tuned filling of trap states. The device acts as an artificial synapse controlled by sub-femtojoule impulses from both the source and gate terminals, consuming lower energy than its biological counterpart. In particular, electrostatic gate modulation, corresponding to biological neuromodulation, additionally regulates the dynamic range and tuning rate of the synaptic weight, independent of the programming (source) impulses. Notably, this heterosynaptic modulation not only improves the learning accuracy and efficiency but also reduces energy consumption in the pattern recognition. Thus, the study presents a new route leading toward the realization of highly networked and energy-efficient neuromorphic electronics.

Coactivator function of positive cofactor 4 (PC4) in Sp1-directed luteinizing hormone receptor (LHR) gene transcription.

The LHR has an essential role in sexual development and reproductive function, and its transcription is subjected to several modes of regulation. In this study, we investigated PC4 coactivator function in the control of LHR transcription. Knockdown of PC4 by siRNA inhibited the LHR basal promoter activity and trichostatin A (TSA)-induced gene transcriptional activation and expression in MCF-7 cells. While overexpression of PC4 alone had no effect on the LHR gene, it significantly enhanced Sp1- but not Sp3-mediated LHR transcriptional activity. PC4 directly interacts with Sp1 at the LHR promoter, and this interaction is negatively regulated by PC4 phosphorylation. The coactivator domain (22-91 aa) of PC4 and DNA binding domain of Sp1 are essential for PC4/Sp1 interaction. ChIP assay revealed significant occupancy of PC4 at the LHR promoter that increased upon TSA treatment. Disruption of PC4 expression significantly reduced TSA-induced recruitment of TFIIB and RNAP II, at the promoter. PC4 functions are beyond TSA-induced phosphatase release, PI3K-mediated Sp1 phosphorylation, and HDAC1/2/mSin3A co-repressor release indicating its role as linker coactivator of Sp1 and the transcriptional machinery. These findings demonstrated a critical aspect of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the human of LHR transcription.

Testis-specific miRNA-469 up-regulated in gonadotropin-regulated testicular RNA helicase (GRTH/DDX25)-null mice silences transition protein 2 and protamine 2 messages at sites within coding region: implications of its role in germ cell development.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTH-null mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.