Structural basis of λCII-dependent transcription activation.

The CII protein of bacteriophage λ activates transcription from the phage promoters PRE, PI, and PAQ by binding to two direct repeats that straddle the promoter -35 element. Although genetic, biochemical, and structural studies have elucidated many aspects of λCII-mediated transcription activation, no precise structure of the transcription machinery in the process is available. Here, we report a 3.1-Å cryo-electron microscopy (cryo-EM) structure of an intact λCII-dependent transcription activation complex (TAC-λCII), which comprises λCII, E. coli RNAP-σ70 holoenzyme, and the phage promoter PRE. The structure reveals the interactions between λCII and the direct repeats responsible for promoter specificity and the interactions between λCII and RNAP α subunit C-terminal domain responsible for transcription activation. We also determined a 3.4-Å cryo-EM structure of an RNAP-promoter open complex (RPo-PRE) from the same dataset. Structural comparison between TAC-λCII and RPo-PRE provides new insights into λCII-dependent transcription activation.

The tigecycline resistance gene tetX has an expensive fitness cost based on increased outer membrane permeability and metabolic burden in Escherichia coli.

Livestock-derived tetX-positive Escherichia coli with tigecycline resistance poses a serious risk to public health. Fitness costs, antibiotic residues, and other tetracycline resistance genes (TRGs) are fundamental in determining the spread of tetX in the environment, but there is a lack of relevant studies. The results of this study showed that both tetO and tetX resulted in reduction in growth and an increased in the metabolic burden of E. coli, but the presence of doxycycline reversed this phenomenon. Moreover, the protection of E. coli growth and metabolism by tetO was superior to that of tetX in the presence of doxycycline, resulting in a much lower competitiveness of tetX-carrying E. coli than tetO-carrying E. coli. The results of RNA-seq showed that the increase in outer membrane proteins (ompC, ompF and ompT) of tetX-carrying E. coli resulted in increased membrane permeability and biofilm formation, which is an important reason for fitness costs. Overall, the increased membrane permeability and metabolic burden of E. coli is the mechanistic basis for the high fitness cost of tetX, and the spread of tetO may limit the spread of tetX. This study provides new insights into the rational use of tetracycline antibiotics to control the spread of tetX.

Serious Risk of Tigecycline Resistance in Escherichia coli Isolated from Swine Manure.

The emergence of the plasmid-mediated tigecycline resistance gene tetX family in pig farms has attracted worldwide attention. The use of tetracycline antibiotics in pig farms has a facilitating effect on the prevalence of the tetX family, but the relationship among its presence, expression, and resistance phenotype in resistant bacteria is unknown. In this study, the presence and expression characteristics of tetracycline resistance genes (TRGs) in 89 strains of doxycycline-resistant E. coli (DRE) isolated from pig manure samples from 20 pig farms under low concentrations of doxycycline stress (2 µg/mL) were analyzed. The detection rate of tetO was 96.63%, which is higher than those of other TRGs, such as tetA (94.38%), tetX (76.40%), tetB (73.03%), and tet(X4) (69.66%). At least three TRG types were present in DRE strains, which thus showed extensive resistance to tetracycline antibiotics, and 37% of these strains were resistant to tigecycline. In the presence of a low concentration of doxycycline, tetA played an important role, and the expression and existence ratio of TRGs indicated low expression of TRGs. Furthermore, the doxycycline resistance of DRE was jointly determined by the total absolute abundance of TRGs, and the absolute abundance of tetX and tet(X4) was significantly positively associated with tigecycline resistance in DRE (P < 0.05). Overall, DRE isolated from swine manure is an important reservoir of the tetX family, which suggests that DRE in swine manure has a high risk of tigecycline resistance, poses a potential threat to human health, and should be of public concern.

Distribution and driving factors of antibiotic resistance genes in treated wastewater from different types of livestock farms.

Treated wastewater from livestock farms is an important reservoir for antibiotic resistance genes (ARGs), and is a main source of ARGs in the environment. However, the distribution and driving factors of ARGs in treated wastewater from different types of livestock farms are rarely reported. In this study, treated wastewater from 69 large-scale livestock farms of different types, including broiler, layer, and pig farms, was collected, and 11 subtypes of ARGs, 2 mobile genetic elements (MGEs) and bacterial community structure were analyzed. The results revealed detection rates of NDM-1 and mcr-1 of 90 % and 43 %, respectively, and the detection rates of other ARGs were 100 %. The relative abundance of ARGs, such as tetA, tetX and strB, in broiler farms was significantly higher than that in layer farms, but the bacterial α diversity was significantly lower than that in other farm types. Furthermore, although the treatment process had a greater impact on the physicochemical properties of the treated wastewater than the livestock type, livestock type was the main factor affecting the bacterial community in the treated wastewater. The analysis of potential host bacteria of ARGs revealed significant differences in the host bacteria of ARGs in treated wastewater from different types of livestock farms. The host bacteria of ARGs in broiler farms mainly belonged to Actinobacteria, layer farms mainly belonged to Proteobacteria, and pig farms mainly belonged to Firmicutes. Additionally, redundancy analysis showed that the distribution of ARGs may have resulted from the combination of multiple driving factors in different types of livestock farms, among which tnpA and NH4+-N were the main influencing factors. This study revealed multiple driving factors for the distribution of typical ARGs in treated wastewater from different types of livestock farms, providing basic data for the prevention and control of ARG pollution in agricultural environments.

Structural basis of AlpA-dependent transcription antitermination.

AlpA positively regulates a programmed cell death pathway linked to the virulence of Pseudomonas aeruginosa by recognizing an AlpA binding element within the promoter, then binding RNA polymerase directly and allowing it to bypass an intrinsic terminator positioned downstream. Here, we report the single-particle cryo-electron microscopy structures of both an AlpA-loading complex and an AlpA-loaded complex. These structures indicate that the C-terminal helix-turn-helix motif of AlpA binds to the AlpA binding element and that the N-terminal segment of AlpA forms a narrow ring inside the RNA exit channel. AlpA was also revealed to render RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Structural analysis predicted that AlpA, 21Q, λQ and 82Q share the same mechanism of transcription antitermination.

Bioconversion of cyanobacteria by black soldier fly larvae (Hermetia illucens L.): Enhancement by antioxidants.

Cyanobacterial blooms have been a global environmental problem for decades. Bioconversion by black soldier fly larvae (BSFL) has been widely reported to be a clean and efficient method to remove organic pollutants. In this study, BSFL bioconversion was used to treat cyanobacterial blooms. Antioxidants (a mixture of l-ascorbic acid [180 mg/kg fresh feed] and α-tocopherol [360 mg/kg fresh feed]) were added to compare bioconversion performance against a non-supplemented group. With increasing proportions of cyanobacteria (0%-25% dry mass), the bioconversion efficiency of the antioxidant group improved significantly compared to the control group, and the survival rate of larvae rose from 96.50-45.50% to 98.00-55.83% with antioxidant addition. The toxic effects of exogenous anti-nutrients could be reduced by the antioxidants through inactivation of trypsin inhibitor and enhancement of the microcystin-LR degradation rate. Overall, the BSFL bioremediation capacity was improved with addition of exogenous antioxidants, verifying both the effects and mechanism of antioxidant addition in promoting the bioconversion of cyanobacteria by BSFL and providing a basis for future application and study.

Structural basis of Mfd-dependent transcription termination.

Mfd-dependent transcription termination plays an important role in transcription-coupled DNA repair, transcription-replication conflict resolution, and antimicrobial resistance development. Despite extensive studies, the molecular mechanism of Mfd-dependent transcription termination in bacteria remains unclear, with several long-standing puzzles. How Mfd is activated by stalled RNA polymerase (RNAP) and how activated Mfd translocates along the DNA are unknown. Here, we report the single-particle cryo-electron microscopy structures of T. thermophilus Mfd-RNAP complex with and without ATPγS. The structures reveal that Mfd undergoes profound conformational changes upon activation, contacts the RNAP ß1 domain and its clamp, and pries open the RNAP clamp. These structures provide a foundation for future studies aimed at dissecting the precise mechanism of Mfd-dependent transcription termination and pave the way for rational drug design targeting Mfd for the purpose of tackling the antimicrobial resistance crisis.

Clinical value of the emergency department in screening and diagnosis of COVID-19 in China.

Since the global outbreak of severe acute respiratory syndrome (SARS) in 2003, China has gradually built a robust prevention and control system for sudden infectious diseases. All large hospitals have a fever clinic that isolates patients with all kinds of acute communicable diseases as the first line of medical defense. The emergency department, as the second line of medical defense in hospitals, is constantly shouldering the heavy responsibility of screening communicable diseases while also treating all kinds of other non-communicable acute and critical diseases (Zhang et al., 2012; Zhu et al., 2015; Wang et al., 2017; Feng et al., 2018; Lu, 2018; Xu and Lu, 2019). An outbreak of pneumonia of unknown etiology that began in Wuhan city (China) has spread rapidly in China since December 2019 (Huang et al., 2020; WHO, 2020; Zhu et al., 2020). In February 2020, the National Health Commission of China named the disease a novel coronavirus pneumonia (NCP); then, it was formally named the coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) on Feb. 11, 2020. The Coronavirus Study Group of the International Committee on Taxonomy of Viruses designated this causative virus as SARS coronavirus 2 (SARS-CoV-2). SARS-CoV-2 belongs to the ß coronavirus genus, and its pathogenic mechanism has not been clarified, which requires further study. To better understand the clinical characteristics of COVID-19 and more effectively prevent and control this disease, we retrospectively analyzed four representative cases of COVID-19 that had recently been screened and diagnosed in our emergency department.

Structural basis of σ appropriation.

Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.

The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis.

Accumulating studies report that microRNAs (miRNAs) are actively involved in skeletal myogenesis. Previously, our study revealed that miR-146b-3p was related to the growth of skeletal muscle. Here, we further report that miR-146b-3p is essential for the proliferation, differentiation, and apoptosis of chicken myoblast. Elevated expression of miR-146b-3p can dramatically suppress proliferation and differentiation, and facilitate apoptosis of chicken myoblast. Besides, we identified two target genes of miR-146b-3p, AKT1 and MDFIC, and found that miR-146b-3p can inhibit the PI3K/AKT pathway. Our study also showed that both AKT1 and MDFIC can promote the proliferation and differentiation while inhibit the apoptosis of myoblast in chicken. Overall, our results demonstrate that miR-146b-3p, directly suppressing PI3K/AKT pathway and MDFIC, acts in the proliferation, differentiation, and apoptosis of myoblast in chicken.

Decreased expression of speckle-type POZ protein for the prediction of poor prognosis in patients with non-small cell lung cancer.

Speckle-type POZ domain protein (SPOP) has been acknowledged as a tumor suppressor gene in numerous types of cancer. However, SPOP expression and its prognostic role in human non-small cell lung cancer (NSCLC) remain unknown. The present study investigated SPOP expression in NSCLC and evaluated its prognostic significance in patients with NSCLC. The results demonstrated that SPOP expression was significantly downregulated in NSCLC tissues at the mRNA and protein level compared with normal lung tissues using reverse transcription-quantitative polymerase chain reaction, and western blot analysis. Immunohistochemical staining results also demonstrated that SPOP was expressed at a low level in 84.1% (132/157) of NSCLC samples and at a high level in 52.2% (12/23) of normal lung samples, whereby the difference was statistically significant (P<0.001). In addition, it was revealed that the level of SPOP was associated with histologic type (P=0.003), tumor differentiation (P=0.046), tumor size (P=0.0036), lymph node metastasis (P=0.041) and clinical stages (P=0.046). Furthermore, the overall survival of patients with high SPOP expression was significantly increased compared with that of patients with low SPOP expression (P=0.003). These results revealed that SPOP expression was downregulated in NSCLC tissues and associated with poor prognosis in patients with NSCLC, suggesting that SPOP is an independent prognostic marker candidate for NSCLC.

Comparison of minocycline and azithromycin for the treatment of mild scrub typhus in northern China.

Scrub typhus, caused by Orientia tsutsugamushi, has recently emerged in northern China where the disease had not been known to exist. Although doxycycline and azithromycin are the recommended agents for the treatment of scrub typhus, clinical responses depend both on the susceptibilities of various O. tsutsugamushi strains and the severity of the disease. A retrospective analysis was conducted on patients diagnosed with mild scrub typhus from August 2013 to January 2016 in the Affiliated Hospital of Nantong University, northern China. A total of 40 patients who received minocycline treatment and 34 patients who received azithromycin treatment were included in the analysis. All patients except one defervesced within 120 h after initiating antimicrobial therapy. Kaplan-Meier curves in association with log-rank test showed that the median time to defervescence was significantly shorter for the minocycline-treated group than the azithromycin-treated group (P = 0.003). There were no serious adverse events during treatment. No relapse occurred in either group during the 1-month follow-up period. In conclusion, both minocycline and azithromycin are effective and safe for the treatment of mild scrub typhus, but minocycline is more active than azithromycin against O. tsutsugamushi infection acquired in northern China.

Coupling of the hydrogen and polyhydroxyalkanoates (PHA) production through anaerobic digestion from Taihu blue algae.

Coupling bio-production of hydrogen and polyhydroxyalkanoates (PHA) from Taihu blue algae through metabolites circulation was investigated. It was found that the pH adjustment, especially basification was more practical and efficient than other methods for the pretreatment of blue algae before anaerobic digestion. On this occasion, SCOD, biogas accumulation and hydrogen content reached 26 mg/gTS, 500 mL and 37.2%, and which were 4.3, 1.3 and 14.4 times of those of the control group, respectively. Secondly, amounts of both butyric acid and hydrogen could be further increased when blue algae was alkali pretreated at pH 13, as the accumulation of butyric acid, acetic acid and hydrogen reached 1.7, 1.4 and 3.8 times compared to those of the control, respectively. Finally, the coupling bio-production of hydrogen and PHA was conducted through pumping organic residues into PHA fermenter from anaerobic digester. Remarkably, it was found that the larger the pumping rate of carbon and nitrogen sources supply, the higher the yield of DCW and PHA could be expected by Bacillus cereus.