Structure-Based Mechanisms of a Molecular RNA Polymerase/Chaperone Machine Required for Ribosome Biosynthesis.

Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.

Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor.

Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the ß subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Microinvasion in hepatocellular carcinoma: predictive factor and application for definition of clinical target volume for radiotherapy.

BACKGROUND: To investigate the correlation between microinvasion and various features of hepatocellular carcinoma (HCC), and to clarify the microinvasion distance from visible HCC lesions to subclinical lesions, so as to provide clinical basis for the expandable boundary of clinical target volume (CTV) from gross tumor volume (GTV) in the radiotherapy of HCC. METHODS: HCC patients underwent hepatectomy of liver cancer in our hospital between July 2019 and November 2021 were enrolled. Data on various features and tumor microinvasion distance were collected. The distribution characteristics of microinvasion distance were analyzed to investigate its potential correlation with various features. Tumor size compared between radiographic and pathologic samples was analyzed to clarify the application of pathologic microinvasion to identify subclinical lesions of radiographic imaging. RESULTS: The average microinvasion distance was 0.6 mm, with 95% patients exhibiting microinvasion distance less than 3.0 mm, and the maximum microinvasion distance was 4.0 mm. A significant correlation was found between microinvasion and liver cirrhosis (P = 0.036), serum albumin level (P = 0.049). Multivariate logistic regression analysis revealed that HCC patients with cirrhosis had a significantly lower risk of microinvasion (OR = 0.09, 95%CI = 0.02 ~ 0.50, P = 0.006). Tumor size was overestimated by 1.6 mm (95%CI=-12.8 ~ 16.0 mm) on radiographic size compared to pathologic size, with a mean %Δsize of 2.96% (95%CI=-0.57%~6.50%). The %Δsize ranged from - 29.03% to 34.78%. CONCLUSIONS: CTV expanding by 5.4 mm from radiographic GTV could include all pathologic microinvasive lesions in the radiotherapy of HCC. Liver cirrhosis was correlated with microinvasion and were independent predictive factor of microinvasion in HCC.

The role of Drp1 - Pink1 - Parkin - mediated mitophagy in perfluorobutane sulfonate- induced hepatocyte damage.

Perfluorobutane sulfonate (PFBS) is recognized as a highly persistent environmental contaminant, notorious for its chemical stability and enduring presence in ecosystems. Its propensity for persistence and environmental mobility allows PFBS to infiltrate the human body, predominantly accumulating in the liver where it poses a potential risk for hepatic damage. This investigation aimed to explore the outcomes of PFBS on the physiological functionalities of hepatocytes in vitro. To this end, hepatocytes were exposed to 750 ug/ml PFBS, followed by an analysis of various cellular phenotypes and functionalities, including assessments of cell viability and mitochondrial integrity. The findings indicated that PFBS exposure led to a suppression of cell proliferation and an increase in apoptotic cell death. Moreover, PFBS exposure was found to augment the generation of reactive oxygen species (ROS) and induce significant mitochondrial dysfunction. Gene expression analysis identified significant changes in genes associated with numerous tumor signaling pathways and autophagy signaling pathways. Further examinations revealed an increase in cellular mitophagy following PFBS exposure, coupled with the activation of the mitophagy-associated Drp1/Pink1/Parkin pathway. Inhibition of mitophagy was observed to concurrently amplify cellular damage and inhibit the Drp1/Pink1/Parkin pathway. Together, these findings highlight PFBS's capacity to inflict hepatocyte injury through mitochondrial disruption, positioning Drp1/Pink1/Parkin-mediated mitophagy as a crucial cellular defense mechanism against PFBS-induced toxicity.

Structural basis for the function of SuhB as a transcription factor in ribosomal RNA synthesis.

Ribosomal RNA synthesis in Escherichia coli involves a transcription complex, in which RNA polymerase is modified by a signal element on the transcript, Nus factors A, B, E and G, ribosomal protein S4 and inositol mono-phosphatase SuhB. This complex is resistant to ρ-dependent termination and facilitates ribosomal RNA folding, maturation and subunit assembly. The functional contributions of SuhB and their structural bases are presently unclear. We show that SuhB directly binds the RNA signal element and the C-terminal AR2 domain of NusA, and we delineate the atomic basis of the latter interaction by macromolecular crystallography. SuhB recruitment to a ribosomal RNA transcription complex depends on the RNA signal element but not on the NusA AR2 domain. SuhB in turn is required for stable integration of the NusB/E dimer into the complex. In vitro transcription assays revealed that SuhB is crucial for delaying or suppressing ρ-dependent termination, that SuhB also can reduce intrinsic termination, and that SuhB-AR2 contacts contribute to these effects. Together, our results reveal functions of SuhB during ribosomal RNA synthesis and delineate some of the underlying molecular interactions.

Changes in Retinal Perfusion in Leber's Hereditary Optic Neuropathy: An Optical Coherence Tomography-Angiography Study.

INTRODUCTION: We aimed to study the change in the retinal perfusion in Leber's hereditary optic neuropathy (LHON). METHODS: Fourteen patients (28 eyes) diagnosed with LHON and 14 healthy controls (28 eyes) were enrolled. The retinal vessel densities in the parafoveal and peripapillary areas were measured with optical coherence tomography-angiography. RESULTS: In the subacute LHON patients, the parafoveal superficial capillary plexus (SCP) and inner retinal thickness (IRT) were significantly reduced in all sectors compared with the controls (all p < 0.05), and the radial peripapillary capillary (RPC) network was significantly reduced in the temporal and inferior temporal sectors compared with the controls (p < 0.05). In the chronic LHON patients, the SCP and IRT were significantly lower in all sectors than in the controls (all p < 0.05), the RPC vessel density and thickness were significantly lower in all sectors than in the controls and lower in the temporal, superior temporal, inferior temporal, and nasal sectors than in the subacute-stage patients (all p < 0.05). CONCLUSION: The retinal structure and the perfusion of the macular and peripapillary areas are reduced in subacute LHON, and the retinal structure and the perfusion of the peripapillary area are further reduced in chronic LHON.

Alterations in the Retinal Vascular Network and Structure in MOG Antibody-Associated Disease: An Optical Coherence Tomography Angiography Study.

BACKGROUND: To determine retinal vessel density in patients with myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). METHODS: Twenty-five patients with MOGAD and 20 healthy participants were enrolled. Patients with MOGAD were divided into myelin oligodendrocyte glycoprotein antibody (MOG-Ab)-positive eyes with a history of optic neuritis (ON; MOG-Ab-ON+ group) or without a history of ON (MOG-Ab-ON- group). Visual function, retinal vessel densities, and thickness were measured. RESULTS: The retinal nerve fiber layer, parafoveal ganglion cell and inner plexiform layers, and vessel densities in the peripapillary and parafoveal areas were significantly decreased in the MOG-Ab-ON+ eyes compared with healthy eyes and MOG-Ab-ON- eyes (all P < 0.05). An increasing number of ON episodes was associated with greater decreases in these variables (all P < 0.05). Visual field mean deviation was not significantly decreased in patients with a history of 1 or 2 episodes of ON, although the relative decreases in retinal nerve fiber layer thickness, parafoveal ganglion cell and inner plexiform layer thickness, peripapillary vessel density, and parafoveal vessel density reached 33.1%, 23.2%, 17.0%, and 11.5% (all P < 0.05), respectively, in eyes with 2 episodes of ON. The mean deviation was significantly correlated with peripapillary vessel density (P < 0.05) after adjustment for other variables. Best-corrected visual acuity was not significantly correlated with optical coherence tomography variables (all P > 0.05). CONCLUSIONS: MOG-Ab-associated ON was associated with significant decreases in retinal structure and vessel density, without significant deteriorations in visual function. The peripapillary vessel density might predict the visual outcomes in patients with MOG-Ab-associated ON.

Synergistic co-regulation and competition by a SOX9-GLI-FOXA phasic transcriptional network coordinate chondrocyte differentiation transitions.

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.

Targeted next-generation sequencing extends the mutational spectrums for OPA1 mutations in Chinese families with optic atrophy.

Purpose: We aim to reveal the disease-causing mutations in 15 Chinese families with optic atrophy (OA). Methods: In total, 15 families with OA were recruited in the present study. Medical histories were carefully reviewed and comprehensive ophthalmic examinations were received by all recruited patients. Targeted next-generation sequencing (NGS) was selectively performed on all probands for mutation detection. Intrafamilial cosegregation and in-silico analyses were subsequently applied to predict the potential pathogenic effects of identified mutations. Results: All included patients presented bilateral vision loss. Their fundus photographs showed temporal or total pallor of the optic discs. Fourteen mutations in the optic atrophy 1 (OPA1) gene were revealed as disease-causing mutations for the 15 families, including eight novel (c.968A>G, c.193C>G, c.1071dupT, c.987_988del, c.2012+2T>G, c.1036-1G>C, c.2126A>G, and c.1036_1038del) and six recurrent (c.1499G>A, c.1800C>A, c.1034G>A, c.2873_2876del, c.112C>T, and c.804_805del) mutations. Conclusions: In conclusion, our study expands the mutational spectrum for the OPA1 gene and implies targeted NGS as an effective approach for the genetic diagnosis of OA, which might help to improve the clinical diagnosis for patients with OA.

Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction.

The transcription factor (TF) SOX18 drives lymphatic vessel development in both embryogenesis and tumour-induced neo-lymphangiogenesis. Genetic disruption of Sox18 in a mouse model protects from tumour metastasis and established the SOX18 protein as a molecular target. Here, we report the crystal structure of the SOX18 DNA binding high-mobility group (HMG) box bound to a DNA element regulating Prox1 transcription. The crystals diffracted to 1.75Å presenting the highest resolution structure of a SOX/DNA complex presently available revealing water structure, structural adjustments at the DNA contact interface and non-canonical conformations of the DNA backbone. To explore alternatives to challenging small molecule approaches for targeting the DNA-binding activity of SOX18, we designed a set of five decoys based on modified Prox1-DNA. Four decoys potently inhibited DNA binding of SOX18 in vitro and did not interact with non-SOX TFs. Serum stability, nuclease resistance and thermal denaturation assays demonstrated that a decoy circularized with a hexaethylene glycol linker and terminal phosphorothioate modifications is most stable. This SOX decoy also interfered with the expression of a luciferase reporter under control of a SOX18-dependent VCAM1 promoter in COS7 cells. Collectively, we propose SOX decoys as potential strategy for inhibiting SOX18 activity to disrupt tumour-induced neo-lymphangiogenesis.

DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors.

Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox-Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.

Synergistic Effect of 2-(Trifluoromethyl) Benzimidazole on the Stability and Performance of Perovskite Solar Cells.

The quality of the perovskite active layer directly impacts the photovoltaic performance of perovskite solar cells (PSCs). Unfortunately, perovskite films produced through solution methods often have a significant number of defects on their surface, which lead to a substantial degradation in the performance of devices. For this reason, a multifunctional additive 2-(trifluoromethyl) benzimidazole (TFMBI) is introduced into perovskite films. Based on the Lewis acid/base coordination principle, the TFMBI double site cooperatively passivates surface defects, inhibiting carrier non-radiative recombination. Simultaneously, the hydrophobic solid group (-CF3) of TFMBI covers the surface, establishing a moisture-oxygen barrier and improving the environmental stability of the devices. In consequence, the power conversion efficiency (PCE) of TFMBI-modified PSCs reached 23.16%, significantly higher than the pristine one with a PCE of 20.62%. Additionally, the unencapsulated target device retained 90.32% of its initial PCE even after being reserved in the air with a relative humidity of 20-30% for 60 days.

Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes.

Organoids with region-specific architecture could facilitate the repair of injuries of the central nervous system. Here we show that human astrocytes can be directly reprogrammed into early neuroectodermal cells via the overexpression of OCT4, the suppression of p53 and the provision of the small molecules CHIR99021, SB431542, RepSox and Y27632. We also report that the activation of signalling mediated by fibroblast growth factor, sonic hedgehog and bone morphogenetic protein 4 in the reprogrammed cells induces them to form spinal-cord organoids with functional neurons specific to the dorsal and ventral domains. In mice with complete spinal-cord injury, organoids transplanted into the lesion differentiated into spinal-cord neurons, which migrated and formed synapses with host neurons. The direct reprogramming of human astrocytes into neurons may pave the way for in vivo neural organogenesis from endogenous astrocytes for the repair of injuries to the central nervous system.

Inflammatory myofibroblastic tumor of the liver following renal transplantation.

Inflammatory myofibroblastic tumor (IMT), previously named inflammatory pseudotumor, is a benign lesion, the exact etiology of which remains obscure; immunosuppression and infections have been speculated to be responsible for the development of pseudotumor. IMT associated with transplantation is rarely reported; we report the first case of IMT of the liver in a renal transplantation patient, who presented with symptoms of abdominal pain. The findings of computed tomography suggested hepatocellular carcinoma or liver abscess, and surgical resection was performed. The lesion was pathologically diagnosed as IMT.

Alterations in the Retinal Vascular Network and Structure in Myelin Oligodendrocyte Glycoprotein Antibody-Associated Optic Neuritis: A Longitudinal OCTA Study.

PURPOSE: To investigate the longitudinal microstructural and microvascular changes in patients with myelin oligodendrocyte glycoprotein antibody-associated optic neuritis (MOG-ON) without new attacks. METHODS: We included 20 eyes of 12 MOG-ON patients without new attacks during the follow-up and 24 eyes of 12 age- and sex-matched healthy controls. RESULTS: The BCVA, retinal vessels and structure were significantly lower in MOG-ON eyes than in healthy eyes(all P < .05). In MOG-ON eyes, the BCVA (p = .408) and mean deviation (p = .854) were not significantly decreased at the follow-up visit. However, there were small, significant decreases in parafoveal vessel density (p = .026), peripapillary vessel density (p = .008), and RNFL thickness (p = .03), but not GCIPL thickness (p = .107). CONCLUSIONS: Ongoing deterioration was observed in RNFL thickness and parafoveal and peripapillary vessel density, but not GCIPL thinning, in MOG-ON eyes without a new attack of ON.

Fluorogenic RNA aptamers to probe transcription initiation and co-transcriptional RNA folding by multi-subunit RNA polymerases.

Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.

Dual-specificity Tyrosine Phosphorylation-regulated Kinase Inhibitor ID-8 Promotes Human Somatic Cell Reprogramming by Activating PDK4 Expression.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) hold great potentials in disease modeling, drug screening and cell therapy. However, efficiency and costs of hiPSCs preparation still need to be improved. METHODS: We screened the compounds that target signaling pathways, epigenetic modifications or metabolic-process regulation to replace the growth factors. After small molecule treatment, TRA-1-60, which is a cell surface antigen expressed by human embryonic stem cells (hESCs), staining was performed to quantify the efficiency of somatic cell reprogramming. Next, small molecule cocktail-induced ESCs or iPSCs were examined with pluripotent markers expression. Finally, Genome-wide gene expression profile was analyzed by RNA-seq to illustrate the mechanism of human somatic cell reprogramming. RESULT: Here, we found that a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of pyruvate dehydrogenase kinase 4 (PDK4) and activation of glycolysis. Furthermore, we identified a novel growth-factor-free hiPSC generation system using small molecules ID-8 (I) and TGFß signal pathway agonist Kartogenin (K). Importantly, we developed IK medium combined with low-dose bFGF to support the long-term expansion of human pluripotent stem cells. IK-iPSCs showed pluripotency and normal karyotype. CONCLUSIONS: Our studies may provide a novel growth-factor-free culture system to facilitate the generation of hiPSCs for multiple applications in regenerative medicine. In Brief Xu et at. found that a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of PDK4 and activation of glycolysis. Furthermore, we established a novel growth-factor-free hiPSC generation system using small molecules ID-8/Kartogenin (IK). IK medium combined with Low-dose bFGF (IKB medium) supported the long-term expansion of human pluripotent stem cells. Highlights ID-8 Enhanced Reprogramming of Human Fibroblasts and Astrocytes Establishment of the Growth-factor-free Reprogramming System Using Small Molecule Compounds IK IKB Medium Maintained the Long-term Expansion of Human Pluripotent Stem Cells ID-8 Promoted Human Somatic Cell Reprogramming by Activating PDK4 Expression.

Donor liver natural killer cells alleviate liver allograft acute rejection in rats.

BACKGROUND: Liver enriched natural killer (NK) cells are of high immune activity. However, the function of donor liver NK cells in allogeneic liver transplantation (LTx) remains unclear. METHODS: Ten Gy of whole body gamma-irradiation (WBI) from a 60Co source at 0.6 Gy/min was used for depleting donor-derived leukocytes, and transfusion of purified liver NK cells isolated from the same type rat as donor (donor type liver NK cells, dtlNKs) through portal vein was performed immediately after grafting the irradiated liver. Post-transplant survival observation on recipients and histopathological detection of liver grafts were adoptive to evaluate the biological impact of donor liver NK cells on recipients' survival in rat LTx. RESULTS: Transfusion of dtlNKs did not shorten the survival time among the recipients of spontaneous tolerance model (BN to LEW rat) after rat LTx, but prolonged the liver graft survival among the recipients depleted of donor-derived leukocytes in the acute rejection model (LEW to BN rat). Compared to the recipients in the groups which received the graft depleted of donor-derived leukocytes, better survival and less damage in the allografts were also found among the recipients in the two different strain combinations of liver allograft due to transfusion of dtlNKs. CONCLUSIONS: Donor liver NK cells alone do not exacerbate liver allograft acute rejection. Conversely, they can alleviate it, and improve the recipients' survival.

[Function of the granaticin biosynthetic gene orf20 from Streptomyces vietnamensis].

OBJECTIVE: The transcription factor SoxR, which serves as a cellular redox sensor, is a global activator of antioxidative stress in Escherichia coli. The granaticin biosynthetic gene orf20 from Streptomyces vietnamensis was found to be a soxR-like gene. This study was carried out towards understanding the physiological function of this gene. METHODS: The orf20 gene was cloned and expressed in E. coli. An orf20 deletion mutant of S. vietnamensis was constructed by using a modified PCR-targeting disruption method. The growth curves of the recombinant E. coli strains and the mutant S. vietnamensis at various paraquat concentrations were assayed to resolve any changes of resistance levels. RESULTS: Soluble expression of orf20 gene in E. coli was achieved, and the recombinant ORF20 protein was tagged by seven histidines at the carboxylic end. An orJ20 deletion mutant of S. vietnamensis DMR20 was successfully constructed. The DMR20 mutant showed no growth changes to the wild, and the ability of sporulation was retained, too. However, the production of granaticin was improved for more than three folds. Compared to the wild type, the DMR20 mutant had no visible changes of resistance to paraquat, however, E. coli carrying the recombinant plasmid pET28b-orf20 received an elevated resistance to paraquat. CONCLUSION: The orf20 gene can complement the soxR gene in E. coli, but is not involved in the regulation of anti-oxidative stress response in S. vietnamensis. Instead, it imposes a negative effect on granaticin production.