Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.

The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.

A bacterial Argonaute from Tepiditoga spiralis with the ability of RNA guided plasmid cleavage.

The eukaryotic Argonaute proteins (eAgos) play an important role in the RNA interference pathway. The function and mechanism of prokaryotic Argonaute proteins (pAgos) in vivo are still unclear although the structure of pAgos and eAgos are highly homologous. Most of the reported pAgos have a preference for 5'P-gDNA, but MpAgo originated from bacteria Marinitoga piezophila preferentially uses 5'OH-gRNA to target DNA and RNA. To enrich our knowledge of this type of Argonaute proteins, here we report an Argonaute protein derived from Tepiditoga spiralis (TsAgo). Like MpAgo, TsAgo has a preference for 5'OH-gRNA. Meanwhile, TsAgo has DNA and RNA cleavage activity in presence of Mn2+ and Mg2+, and TsAgo has catalytic activity at 37-70 °C. In addition, TsAgo can tolerate mismatches in the 5'-end and 3'-tail regions of guides but is sensitive to mismatches in the 5'-seed and central regions of guides, especially the central region. Furthermore, the EMSA assay reveals that TsAgo exhibits a stronger binding affinity for 5'OH-gRNA than 5'P-gRNA which is consistent with its cleavage activity. Moreover, the structural modeling analysis demonstrates that like MpAgo, TsAgo has an ordered α5 at the C terminus of the PIWI domain which may hinder to binding of 5' phosphate. Importantly, we find that TsAgo can target and cut plasmid DNA in vitro at 60 °C under the direction of RNA guides. These studies broaden our understanding of pAgos, and demonstrate that TsAgo can be regarded as an RNA-guided programmable nuclease for cleaving plasmids.

Bacteria-Mediated Tumor Therapy via Photothermally-Programmed Cytolysin A Expression.

By leveraging the ability of bacteria to express therapeutic protein cytolysin A (ClyA) through plasmid transformation, a thermally-activated biohybrid (TAB@Au) is constructed by biomineralizing gold nanoparticles (AuNPs) on the E. coli surface. Due to the feature of anaerobic bacteria homing to tumor microenvironments, the bacteria-based antitumor vehicles can be efficaciously accumulated at tumor sites. Under NIR laser irradiation, the biomineralized AuNPs harvest transdermal photons and convert them into local heat for photothermal therapy. After that, the produced heat elicits the expression of ClyA for killing tumor cells. In vitro and in vivo experiments verify the conception that the current therapeutic modality greatly inhibits the proliferation of tumor cells. In terms of the spatial specificity and non-invasiveness of NIR laser, the bacteria-based phototherapy represents an appealing way for tumor therapy.

The T helper type 17/regulatory T cell imbalance was associated with Ras-GTPase overexpression in patients with pulmonary hypertension associated with chronic obstructive pulmonary disease.

Pulmonary hypertension (PH) is a common but dangerous complication in chronic obstructive pulmonary disease (COPD). We hypothesized that dysregulation in the T helper type 17 (Th17) compartment could contribute to the development of COPD-associated PH (COPD-PH). To investigate this hypothesis, patients with COPD-PH and age- and sex-matched healthy controls were recruited, and their circulating CD4+ T cells were activated using anti-CD3/CD28 antibodies. The frequency of interleukin-17 (IL-17) -secreting cells was significantly higher in COPD-PH patients than in healthy controls. The secretion of IL-17 was significantly higher from COPD-PH CD4+ T cells than from control CD4+ T cells, whereas the secretion of interferon-γ and IL-4 was not significantly different. The expression of transforming growth factor-ß, on the other hand, was significantly higher in healthy controls than in COPD-PH patients. Activated CD4+ T cells from COPD-PH patients also presented significantly lower forkhead box P3 (FOXP3) and higher retinoic acid receptor-related orphan C2 (RORC2) expression than CD4+ T cells from healthy controls. In both controls and patients, a negative correlation between RORC2 and FOXP3 was found, ex vivo and after CD3/CD28 activation. The serum IL-6 level was slightly higher in COPD-PH patients than in controls, but the IL-6 transcription by monocytes was comparable in COPD-PH patients and controls. Interestingly, CD4+ T cells from COPD-PH patients presented significantly higher levels of Kirsten rat sarcoma viral oncogene homolog and neuroblastoma RAS viral oncogene homolog than CD4+ T cells from healthy controls. Inhibiting Ras-GTPases using farnesylthiosalicylic acid significantly reduced the ratio of RORC2/FOXP3 expression in CD4+ T cells. Overall, we demonstrated that an imbalance of Th17/regulatory T cells was a hallmark of COPD-PH.

An upregulation of CD8+CD25+Foxp3+ T cells with suppressive function through interleukin 2 pathway in pulmonary arterial hypertension.

Accumulating evidence suggests that abnormal inflammation plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH). CD8+CD25+Foxp3+ T cell is a novel cell subtype, and its role in PAH is not yet investigated. Here, we observed that PAH patients presented a significant upregulation of CD8+CD25+Foxp3+ T cells and a downregulation of CD4+CD25+Foxp3+ T cells compared to healthy controls. Regardless, the total number of CD25+Foxp3+ T cells in PAH patients was still smaller than that in healthy controls. Compared to CD8+CD25- T cells, CD8+CD25+ T cells presented higher Foxp3 expression, lower interferon (IFN)-γ expression and higher transforming growth factor (TGF)-ß expression, in both healthy and PAH individuals. The CD8+CD25+ T cells in PAH patients also demonstrated regulatory function by suppressing the proliferation of CD4+CD25- and CD8+CD25- effector T cells, albeit at lower efficiency than CD4+CD25+ T cells from PAH patients and healthy volunteers. CD8+CD25+ T cells from PAH responded to interleukin (IL)-2 supplement by expansion and upregulating Foxp3 expression. In PAH patients, IL-2-treated CD8+CD25+ T cells were more potent at inhibiting CD4+CD25- effector T cell proliferation than IL-2-untreated CD8+CD25+ T cells. Together, we found an upregulation of CD8+CD25+Foxp3+ T cells in PAH patients, and this T cell population presented suppressive activity that could be enhanced by IL-2 treatment.

Nocturnal blood pressure fluctuation and associated influential factors in severe obstructive sleep apnea patients with hypertension.

PURPOSE: Obstructive sleep apnea syndrome (OSAS) can induce dramatic blood pressure (BP) fluctuations during sleep and it can be associated with hypertension. We investigated the properties and associated influential factors of BP fluctuation in severe OSAS with and without hypertension. METHODS: Two hundred one severe OSAS subjects were divided into hypertensive and normotensive groups. BP was continuously monitored via measurement of pulse transmit time (PTT). The value of apnea-related systolic BP elevation (ΔSBP) was used to reflect the amplitude of BP fluctuation, and the SBP index (the number of ΔSBP > 10 mmHg per hour of sleep time) was used to stand for the frequency of significant BP fluctuations. RESULTS: Compared with the normotensive group, △SBP and SBP index were higher in the hypertensive group (13.8 ± 4.4 mmHg vs 10.9 ± 3.1 mmHg; 44.8 ± 21.3 events/h vs 26.8 ± 15.8 events/h, all p < 0.001). Multiple regression analysis showed that percentage of sleep time with oxygen saturation < 90% (TST90) and SBP index correlated more with mean level of awakeness and sleep SBP than with apnea-hypopnea index (AHI). Analysis of all apnea events demonstrated that △SBP and the frequency of BP fluctuations were more remarkable following hypoxia than following arousal; △SBP correlated more with oxygen desaturation degree (r = 0.388, p < 0.01) and minimal SpO2 (r = 0.392, p < 0.01) than with apnea length and desaturation duration. CONCLUSIONS: In severe OSAS, nocturnal and awake BP levels are associated more with the nocturnal hypoxic duration and BP fluctuation than with AHI. Nocturnal BP fluctuation can be induced by both hypoxia and arousal, and especially by hypoxia. TRIAL REGISTRATION: NCT02876471.

Gestation-specific changes in maternal thyroglobulin during pregnancy and lactation in an iodine-sufficient region in China: a longitudinal study.

OBJECTIVES: To describe the changes in thyroglobulin (Tg) based upon gestational and postpartum concentrations in healthy pregnant women from an iodine-sufficient region in China, and to evaluate the use of Tg as a biomarker for iodine-sufficient pregnant women. DESIGN: A longitudinal study of Tg change in normal pregnant women from an iodine-sufficient region. PATIENTS AND MEASUREMENTS: Blood and urine samples were obtained from 133 pregnant women. Urinary iodine concentration (UIC) was measured using an ammonium persulfate method. Serum iodine concentration was required by inductively coupled plasma mass spectrometry (ICP-MS). Serum thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), total thyroxine (TT4), total triiodothyronine (TT3), antithyroid peroxidase antibody (TPOAb), antithyroglobulin antibody (TgAb) and Tg were measured using an electrochemiluminescence immunoassay. RESULTS: Thyroglobulin concentrations were higher in early pregnancy (pregnancy at 8 weeks vs nonpregnancy: 11·42 ng/ml vs 8·8 ng/ml, P < 0·01) and maintained a stable level, and then increased greatly at the 36th week. After delivery, Tg decreased to nonpregnant levels. During pregnancy, maternal Tg was not correlated with thyroid function, UIC or urine iodine-creatinine ratio (UI/Cr). Cord blood Tg was much higher compared to maternal Tg levels at the 36w (57·34 vs 14·86 ng/ml, P < 0·001) and correlated positively with cord FT4 (r = 0·256, P < 0·05), cord TT4 (r = 0·263, P < 0·05) and maternal UI/Cr at 36w (r = -0·214, P < 0·05). CONCLUSIONS: Our work demonstrates that Tg is elevated during pregnancy, and the effect of pregnancy should be taken into consideration when Tg is used as a biomarker for the iodine status. Cord blood Tg is much higher than maternal Tg levels at the 36w and is correlated with maternal iodine status.

Improvement of PbSn Solder Reliability with Ge Microalloying-Induced Optimization of Intermetallic Compounds Growth.

PbSn solders are used in semiconductor devices for aerospace or military purposes with high levels of reliability requirements. Microalloying has been widely adopted to improve the reliability for Pb-free solders, but its application in PbSn solders is scarce. In this article, the optimization of PbSn solder reliability with Ge microalloying was investigated using both experimental and calculation methods. Intermetallic compounds (IMC) growth and morphologies evolution during reliability tests were considered to be the main factors of device failure. Through first-principle calculation, the growth mechanism of interfacial Ni3Sn4 was discussed, including the formation of vacancies, the Ni-vacancies exchange diffusion and the dominant Ni diffusion along the [1 0 0] direction. The doping of Ge in the cell increased the exchange energy barrier and thus inhibited the IMC development and coarsening trend. In three reliability tests, only 0.013 wt% Ge microalloying in Pb60Sn40 was able to reduce IMC thickness by an increment of 22.6~38.7%. The proposed Ge microalloying method in traditional PbSn solder could yield a prospective candidate for highly reliable applications.

Effect of Sb Content on the Microstructure and Mechanical Properties of Eutectic SnPb Solder.

SnPb solder was widely used in electronic packaging for aerospace devices due to its high reliability. However, its creep resistance is poor and can be improved by adding alloying elements. The effects of Sb content on the microstructure, tensile, and creep properties of eutectic SnPb solder were investigated. Sb addition effectively improved the mechanical properties of the SnPb solder. When Sb content exceeds 1.7 wt.%, SbSn intermetallic compounds (IMCs) occurred. And increasing the Sb content increased the tensile strength. Furthermore, Sb addition decreased the steady-state creep rate and increased the stress exponent n, suggesting that the creep resistance had been enhanced, which may be attributed to the hindrance of dislocation movement by SbSn IMCs, as well as the reduction in phase boundaries, which consequently reduced grain boundary sliding.

EBUS-TBLC increase the diagnosis rate in different type of peripheral pulmonary lesions.

Background and objective: Recently, endobronchial ultrasonography with guide sheath-guided (EBUS-GS) has been increasingly used in the diagnosis of peripheral pulmonary lesions (PPLs) from human natural orifice. However, the diagnostic rate is still largely dependent on the location of the lesion and the probe. Here, we reported a new procedure to improve the diagnostic rate of EBUS-transbronchial lung cryobiopsy (EBUS-TBLC), which performed under general anesthesia with laryngeal mask airway (LMA) in all of the patients. This study retrospectively evaluated the diagnosis of PPLs with 'blind-ending' type (Type I) and 'pass-through' type procedures (Type II) of EBUS-GS-TBLB or EBUS-TBLC respectively. Methods: Retrospective review of 136 cases performed by EBUS-GS-TBLB or EBUS-TBLC for PPLs over 2 years. Results: A total of 126 cases EBUS-GS-TBLB or EBUS-TBLC were performed during the study period. Among them, 66 (52.4%) were performed Type I and 60 (47.6%) were performed Type II. Clinical baseline characteristics did not differ between two groups. The overall diagnosis rate of 126 patients with EBUS-GS-TBLB or EBUS-TBLC was 73% (92/126), and different method type have significant influence on the diagnostic yield (P = 0.012, x2 = 4.699). Among them, diagnostic yields for Type I with forceps biopsy (n=34), Type I with cryobiopsy (n=32), Type II with forceps biopsy (n=30), and Type II with cryobiopsy (n=30) were 72.5%, 64.5%, 70.4% and 74.2% respectively (Figure 2A). The study further compared the outcomes of different procedures in concentric and eccentric lesion. Diagnostic yields for Type I with eccentric (n=30), Type I with concentric (n=36), Type II with eccentric (n=34), and Type II with concentric (n=26) were 58.2%, 76.9%, 60.2% and 74.8%, respectively (P < 0.05). The incidence of complications in 126 patients was 2.6%. Conclusion: EBUS-GS-TBLB and EBUS-TBLC both are very safe and highly diagnostic technique; different method types have significant influence on the diagnostic yield. Moreover, Type II procedure has higher diagnostic yield. In addition, Type I with eccentric had the lowest diagnosis yield.

Identification of key biomarkers in ischemic stroke: single-cell sequencing and weighted co-expression network analysis.

PURPOSE: At present, there is a lack of accurate early diagnostic markers for ischemic stroke. METHODS: By using dimensionality reduction cluster analysis, differential expression analysis, weighted co-expression network analysis, protein-protein interaction network analysis, cell heterogeneity and key pathogenic genes were identified in ischemic stroke. Immunomicroenvironment analysis was used to explore the immune landscape and immune associations of key genes in ischemic stroke. The analysis platform we use is R software (version 4.0.5). PCR experiments were used to verify the expression of key genes. RESULTS: Single cell sequencing data in ischemic stroke can be annotated as fibroblast cells, pre-B cell CD34, neutrophils cells, bone marrow (BM), keratinocytes, macrophage, neurons and mesenchymal stem cells (MSC). By the intersection of differential expression analysis and WGCNA analysis, 385 genes were obtained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes were highly correlated with multiple functions and pathways. Protein-protein interaction network analysis revealed that MRPS11 and MRPS12 were key genes, both of which were down-regulated in ischemic stroke. The Pseudo-time series analysis found that the expression of MRPS12 decreased gradually with the differentiation of pre-B cell CD34 cells in ischemic stroke, suggesting that the downregulation of MRPS12 expression may play an important role in ischemic stroke. At last, PCR showed that MRPS11 and MRPS12 were significantly down-regulated in peripheral blood of patients with ischemic stroke. CONCLUSIONS: Our study provides a reference for the study of pathogenesis and key targets of ischemic stroke.

New perspective on exploring the predictive factors of blood pressure reduction during CPAP treatment in people with severe OSA and hypertension: a prospective observational study.

BACKGROUND: The predictive factors of blood pressure (BP) response to continuous positive airway pressure (CPAP) in obstructive sleep apnoea (OSA) are still being explored. We aimed to assess the antihypertensive effect of CPAP considering the obstructive respiratory event-triggered BP surge profiles in 130 subjects with severe OSA and untreated hypertension. METHODS: Nocturnal BP was monitored continuously and synchronised with polysomnography. Event-triggered BP surge profiles were studied: BP surge as the value of event-related systolic BP (SBP) elevation; BP index as the number of BP surge events of ≥10 mm Hg per hour. Patients were then divided into two groups according to the median BP index (high and low BP surge groups) and assigned to 4 weeks of CPAP. Changes in BPs and plasma biomarkers were compared. After the initial evaluation, patients with a better BP response in the high BP surge group were then followed up for the second evaluation at 24 months. RESULTS: Overall, a modest decrease was observed in both office and asleep BPs at the 4-week follow-up; however, BPs dropped more markedly in patients in the high BP surge group than those in the low BP surge group, in both office SBP (5.3 mm Hg vs 2.2 mm Hg, p=0.003) and diastolic BP (4.0 mm Hg vs 1.2 mm Hg, p<0.001), especially the asleep SBP (9.0 mm Hg vs 2.1 mm Hg, p<0.001). For 30 cases in the high BP surge group, optimal BP control was achieved in 60.0% of patients and BP<140/90 mm Hg reached up to 83.3% after 24 months of CPAP. Linear regression revealed that BP index was significantly associated with BP decrease during CPAP treatment. CONCLUSIONS: Our results suggested that high event-triggered BP surge was a sensitive predictor of BP response to CPAP in patients with severe OSA and untreated hypertension. TRIAL REGISTRATION NUMBER: Clinical Trials.gov Identifier: NCT03246022; https://clinicaltrials.gov/ct2/show/NCT03246022?term=NCT+03246022&draw=2&rank=1.

Gestational and Postpartum Complications in Patients with First Trimester Thyrotoxicosis: A Prospective Multicenter Cohort Study from Northeast China.

Objective: Gestational transient thyrotoxicosis (GTT) and Graves' disease (GD) are the most common causes of hyperthyroidism during pregnancy. However, few studies have compared pregnancy outcomes of patients who had GTT with those who had GD in the first trimester of pregnancy. Methods: We conducted a prospective multicenter cohort study in China. Participants received questionnaires, physical examinations, and underwent measurements of thyrotropin (TSH), free thyroxine (fT4), thyroid peroxidase antibody (TPOAb), TSH receptor antibody (TRAb), and urinary iodine in the first trimester. The patients diagnosed with either GTT or GD and normal thyroid function (NTF) group were followed until delivery. The thyroid function and pregnancy outcomes were reported. Results: A total of 125 pregnant women with thyrotoxicosis and 246 age-matched pregnant women with NTF were included. (1) The thyroid function of the GTT group returned to normal range in the third trimester, but was consistently abnormal in the GD group. (2) The incidence of gestational diabetes mellitus (GDM) in the GTT group (11.5%, 9/78) was significantly higher than that in NTF group (4.9%, 12/246) (p = 0.037). The incidence of premature delivery in the GD untreated (30.8%, 8/26, p = 0.002) and treated groups (28.6%, 6/21, p = 0.008) was both, respectively, higher than that in the NTF group (7.7%, 19/246). Miscarriage (15.4%, 4/26 vs. 3.7%, 9/246, p = 0.026) and gestational hypertension (19.2%, 5/26 vs. 3.3%, 8/246, p = 0.004) were more prevalent in the GD untreated group than in the NTF group. (3) The presence of positive TRAb and positive TPOAb in the first trimester were independent risk factors for miscarriage (odds ratio [OR] = 5.23, confidence interval [CI] = 1.11-24.78, p = 0.037) and low birth weight infants (OR = 7.76, CI = 1.23-48.86, p = 0.029), respectively. Conclusion: In conclusion, pregnancy outcomes appear variable, according to the etiology of first trimester thyrotoxicosis. GTT appears to be associated with GDM. GD appears to be associated with an increased risk of premature delivery, gestational hypertension, and miscarriage. The diagnosis of GTT and GD patients during early pregnancy and appropriate treatment of GD patients may be associated with improved pregnancy outcomes.

The impact of global, regional, and national population ageing on disability-adjusted life years and deaths associated with diabetes during 1990-2019: A global decomposition analysis.

AIMS: To understanding the net regional, national, and economic effect of global population ageing on diabetes and its trends during 1990 and 2019 worldwide. METHODS: We employed a decomposition method to estimate the impact of population ageing on diabetes-related disability-adjusted life years (DALYs) and total deaths in 204 countries from 1990 to 2019 at the global, regional, and national level. This method separated the net effect of population ageing from population growth and changes in mortality. RESULTS: Globally, population ageing has become the major contributor to diabetes-related deaths since 2013. The increases in diabetes-related deaths attributed to population ageing exceeding the decreases in mortality change. Population ageing produced an additional 0.42 million diabetes-related deaths and 14.95 million DALYs from 1990 to 2019. At the regional level, population ageing is associated with the increases in diabetes-related deaths in 18 out of 22 regions. The highest increase in diabetes-related deaths attributed to population ageing occurred in men in East Asia (136.31%) and women in Central Latin America (118.58%). The proportion of diabetes-related deaths and DALYs attributable to population ageing showed a bell-shaped relationship with sociodemographic index (SDI) and peaked at high-middle-SDI countries. CONCLUSIONS: The decreases in diabetes-related deaths attributed to mortality change exceeded the increases attributed to population ageing between 1990 and 2019 globally and regionally. The diabetes-related deaths in high-middle-SDI countries were most impacted by population ageing.

Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production.

Adenosine triphosphate (ATP), as a universal energy currency, takes a central role in many biochemical reactions with potential for the synthesis of numerous high-value products. However, the high cost of ATP limits industrial ATP-dependent enzyme-catalyzed reactions. Here, we investigated the effect of cell-surface display of phosphotransferase on ATP regeneration in recombinant Escherichia coli. By N-terminal fusion of the super-folder green fluorescent protein (sfGFP), we successfully displayed the phosphotransferase of Pseudomonas brassicacearum (PAP-Pb) on the surface of E. coli cells. The catalytic activity of sfGFP-PAP-Pb intact cells was 2.12 and 1.47 times higher than that of PAP-Pb intact cells, when the substrate was AMP and ADP, respectively. The conversion of ATP from AMP or ADP were up to 97.5% and 80.1% respectively when catalyzed by the surface-displayed enzyme at 37 °C for only 20 min. The whole-cell catalyst was very stable, and the enzyme activity of the whole cell was maintained above 40% after 40 rounds of recovery. Under this condition, 49.01 mg/mL (96.66 mM) ATP was accumulated for multi-rounds reaction. This ATP regeneration system has the characteristics of low cost, long lifetime, flexible compatibility, and great robustness.

CHK2 Promotes Metabolic Stress-Induced Autophagy through ULK1 Phosphorylation.

Reactive oxygen species (ROS) act as a signaling intermediate to promote cellular adaptation to maintain homeostasis by regulating autophagy during pathophysiological stress. However, the mechanism by which ROS promotes autophagy is still largely unknown. Here, we show that the ATM/CHK2/ULK1 axis initiates autophagy to maintain cellular homeostasis by sensing ROS signaling under metabolic stress. We report that ULK1 is a physiological substrate of CHK2, and that the binding of CHK2 to ULK1 depends on the ROS signal and the phosphorylation of threonine 68 of CHK2 under metabolic stress. Further, CHK2 phosphorylates ULK1 on serine 556, and this phosphorylation is dependent on the ATM/CHK2 signaling pathway. CHK2-mediated phosphorylation of ULK1 promotes autophagic flux and inhibits apoptosis induced by metabolic stress. Taken together, these results demonstrate that the ATM/CHK2/ULK1 axis initiates an autophagic adaptive response by sensing ROS, and it protects cells from metabolic stress-induced cellular damage.

Transplantion of predominant Lactobacilli from native hens to commercial hens could indirectly regulate their ISC activity by improving intestinal microbiota.

In poultry, HyLine (HL) Hens are known for their excellent laying performance. However, ZhenNing (ZN) Hens, a native chicken breed in China, are known for their unique flavour. The intestinal mucosa, which is the main organ for nutrient absorption, could affect livestock product quality. In ZN Hens' intestinal mucosa, we found more villus wrinkles, larger villus circumference and higher amino acid transporters mRNA abundance compared with HL Hens. Among three laying periods of ZN Hens, in the intestinal lumen, Lactobacillus salivarius (L. sa.), Lactobacillus agilis (L. ag.) and Lactobacillus aviarius were the predominant species in the laying peak period. Furthermore, multiple-antibiotics feeding in ZN Hens and predominant Lactobacillus feeding in HL Hens suggested that these Lactobacilli could indeed increase villus wrinkles and improve intestinal absorption. In HL Hens, L. sa. + L. ag. treatment could promote organoids budding in vitro, and promote epithelial proliferation in vivo. Collectively, the unique intestinal mucosa morphology in ZN Hens was due to the high abundance of intestinal L. sa. and L. ag. Transplant these Lactobacilli to HL Hens could increase their intestinal probiotics abundance, fine adjust the intestinal stem cell function and promote the epithelial proliferation, in turn, increase villus winkles and mucosal absorption area.

Prototheca bovis induces autophagy in bovine mammary epithelial cells via the HIF-1α and AMPKα/ULK1 pathway.

Prototheca bovis, a highly contagious pathogen, causes bovine mastitis, resulting in premature culling of affected cows and severe economic losses. Infection with P. bovis caused oxidative stress and apoptosis in bovine mammary epithelial cells (bMECs); however, mechanisms underlying P. bovis-induced autophagy remain unclear. Therefore, the autophagy flux induced by P. bovis in bMECs was analyzed by Western blot and laser scanning confocal microscopy. Expression levels of proteins in the HIF-1α and AMPKα/ULK1 pathway, including HIF-1α, AMPKα, p-AMPKα, ULK1, p-ULK1, mTOR, and p-mTOR, plus expression of autophagy-related genes including SQSTM1/p62, Atg5, Beclin1, and LC3II/LC3I, were quantified with Western blot. Infection with P. bovis induced autophagosomes and LC3 puncta in bMECs that were detected using transmission electron microscopy and laser scanning confocal microscopy, respectively. In addition, lysosome-associated proteins Rab7 and LAMP2a, and lysosomal activity were measured with Western blot and laser scanning confocal microscopy. Infection with P. bovis induced an unobstructed autophagic flux, increased protein expression of LC3II/LC3I, and decreased SQSTM1/p62 protein expression at 6 hpi. Furthermore, P. bovis upregulated protein expression in the HIF-1α and AMPKα/ULK1 pathway and increased the ratio of LC3II/LC3I, implying autophagy was activated in bMECs. However, deletion of AMPKα or ULK1 decreased LC3II/LC3I expression levels and LC3 puncta numbers, suggesting that autophagy was inhibited in bMECs. Additionally, deficiency of HIF-1α decreased protein expression of AMPKα and ULK1 as well as LC3 puncta numbers, and autophagy induced by P. bovis was also inhibited in bMECs. At 6 hpi, lysosome-associated protein Rab7 was decreased and LAMP2a was increased, indicating normal autophagy. In contrast, at 12 hpi, expression of Rab7 and LAMP2a proteins indicated that autophagy was inhibited in bMECs at that time. Therefore, we confirmed that P. bovis infection induced autophagy in bMECs via the HIF-1α and AMPKα/ULK1 pathway, with involvement of lysosome-associated protein Rab7 and LAMP2a.

A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA.

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.

Co-overexpression of RIOK1 and AKT1 as a prognostic risk factor in glioma.

Glioblastoma multiforme (GBM) is one of the most frequent primary malignancies of the brain. Although the treatment strategy has significantly improved, patient prognosis remains poor. In vitro studies have shown that the right open reading frame kinase 1/protein kinase B (RIOK1-AKT) signaling pathway plays an important role in the malignant phenotype of glioma cells. This study aimed to investigate the co-expression of RIOK1 and ATK in glioma tissues and its clinical significance. Compared with normal tissues, RIOK1 and AKT1 expression were significantly upregulated in glioma tissues. In addition, patients with higher World Health Organization staging grades had increased RIOK1 and AKT1 expression levels, and RIOK1 and AKT1 expression were positively correlated. Notably, both RIOK1 and AKT1 expressions were correlated with poor prognosis. In vitro experiments showed that silencing RIOK1 inhibited the proliferation, migration, and invasion of glioma cell lines by suppressing AKT and c-Myc expression. These results indicate that the RIOK1-AKT1 axis could play an important role in GBM progression.