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Linalool, as a fragrant monoterpene, is an important feedstock for food, pharmaceuticals, and cosmetics industries. Although our previous study had significantly increased linalool production by the directed evolution of linalool synthase and overexpression of the whole mevalonate pathway genes, the engineered yeast strain suffered from dramatically reduced biomass. Herein, a stress-free linalool-producing yeast cell factory was constructed by the combinational regulation of linalool synthase and farnesyl diphosphate synthase instead of multienzyme overexpression. First, the expression level of linalool synthase was successfully enhanced by introducing a N-terminal SKIK tag, which improved linalool production by 3.3-fold. Subsequently, the modular assembly of linalool synthase and dominant negative farnesyl diphosphate synthase via short peptide tags efficiently converted geranyl pyrophosphate to linalool. Additional downregulation of the native farnesyl diphosphate synthase led to the highest reported linalool production (80.9 mg/L) in yeast. This combinatorial modulation strategy may also be applied to the production of other high-value monoterpenes.

We describe 3 similar cases of rickettsial disease that occurred after tick bites in a mountainous rural area of Shandong Province, China. Next-generation sequencing indicated the etiologic agent of 1 patient was Rickettsia conorii subspecies indica. This agent may be more widely distributed across China than previously thought.

Background Brucellosis is endemic in many areas in China. The current diagnosis of Brucellosis predominantly relies on the traditional bacterial culture and serum agglutination test. In this study, we aimed to explore the value of ELISA in the diagnosis of Brucellosis in Chinese population. Methods We recruited 235 patients with a diagnosis of Brucellosis at different clinical stages: 117 in acute, 78 in subacute, and 40 in chronic. We also recruited 248 control patients who presented with similar clinical symptoms but with a different diagnosis other than Brucellosis. In addition, 90 healthy volunteers were also recruited. Bacterial culture, agglutination test and ELISA assay were performed to detect Brucella spp. Results Among 235 patients with Brucellosis, 51 (21.7%) was positive for bacterial culture, 150 (63.8%) were positive by agglutination test, and 232 (98.7%) were positive by ELISA (IgG and/or IgM). When we stratified the patients based on the disease stages (acute, subacute and chronic), ELISA was the most sensitive method and showed a highest positive rate in all stages. By Receiver Operating Characteristic Curve analysis of ELISA results, we found that measurement of IgG level was superior to measurement of IgM level (AUC, 0.993 versus 0.877). Since the measurement of IgG itself missed rare cases in acute phase, we recommended measuring IgG and IgM simultaneously by ELISA for the diagnosis of Brucellosis. In term of the specificity of ELISA in the diagnosis of Brucellosis, our study showed that only 1.6% (4/248) non-Brucellosis patients were positive by ELISA; all positive cases were IgM only and none showed positive IgG. Similar results were found in healthy volunteers. In summary, our study concluded that ELISA is the most sensitive and specific method to detect Brucellosis in Chinese population. Conclusions ELISA assay is sensitive, fast, and convenient to detect Brucellosis. It shows the high sensitivity and specifity and should be used as a routine lab test when Brucellosis is suspected in clinical practice.

Type I interferon (IFN) response is central for host defense against viral infection. Tripartite motif 27 (TRIM27) is implicated in antiviral innate immune response; however, whether it affects the replication of hepatitis C virus (HCV) and the underlying mechanisms remain uncharacterized. Here, we show that TRIM27 expression is induced in Huh7.5 human hepatoma cells infected with HCV or stimulated with type I IFNs in vitro. In addition, TRIM27 overexpression increases and its knockdown decreases viral RNA and protein levels, suggesting that TRIM27 positively regulates HCV replication. Mechanistically, TRIM27 inhibits type I IFN response against HCV infection through inhibiting IRF3 and NF-κB pathways, since TRIM27 mutant unable to inhibit these two inflammatory pathways fails to promote HCV replication. Taken together, this study identifies TRIM27 as a novel positive regulator of HCV replication, and also implicates that targeting TRIM27 may serve as a therapeutic strategy for controlling HCV replication.

Background Brucellosis is a common zoonotic disease that is prevalent in many areas worldwide. This infectious disease can occasionally affect the central nervous system but intracranial arteries are rarely involved. Case presentation A 17-year-old female who had a history of recurrent fever for 1 month was admitted for subarachnoid hemorrhage due to cerebral aneurysm rupture. Surgery was performed to fix the aneurysm, but the patient had persistent fever after the surgery. Cerebrospinal fluid testing showed a high white blood cell count and elevated protein level but no pathogen was identified in the first two tests. Brucella melitensis was identified in the third cerebrospinal fluid culture, and a diagnosis of brucellosis was finally rendered. The patient was subsequently treated with anti-Brucella medications and her symptoms improved significantly at the last follow-up. Conclusion Although extremely rare, Brucella-induced cerebral aneurysms can occur and this should be considered in the differential diagnosis of cerebrovascular accidents, especially in Brucella epidemic areas.

For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRISPR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. This results in impaired polystyrene bead binding and transport but not gliding of cells on solid surfaces. Notably, assembly, intraflagellar transport, and protein import into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.

Background As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported sporadically for biosynthesis of caffeic acid via free plasmid‑mediated pathway assembly. However, the production was far from satisfactory and even relied on the addition of precursor. Results In this study, we first established a controllable caffeic acid pathway by employing a modified GAL regulatory system in S. cerevisiae and realized de novo biosynthesis of 313.8 mg/L caffeic acid from glucose. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways and overexpressing rate-limiting enzymes led to about 2.5-fold improvement in the caffeic acid production, reaching up to 769.3 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid de novo synthesized by engineered yeast. Conclusions Caffeic acid production in S. cerevisiae strain was successfully improved by adopting a glucose-regulated GAL system and comprehensive metabolic engineering strategies. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites.

For the unicellular algaChlamydomonas reinhardtii, the presence ofN-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. It is unknown whether the composition ofN-glycans attached to respective proteins is important for these processes. To this end, we examined severalC. reinhardtiiinsertional mutants and a CRIPSR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing alteredN-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction inN-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. In addition, polystyrene bead binding and transport is impaired. Notably, assembly, Intraflagellar Transport and FMG-1B transport into flagella are not affected by alteredN-glycosylation. Thus, we conclude that properN-glycosylation of flagellar proteins is crucial for adheringC. reinhardtiicells onto surfaces, indicating thatN-glycans mediate surface adhesion via direct surface contact.

At present, only little is known about the enzymatic machinery required for N-glycosylation in Chlamydomonas reinhardtii, leading to the formation of N-glycans harboring Xyl and methylated Man. This machinery possesses new enzymatic features, as C. reinhardtii N-glycans are independent of β1,2-N-acetylglucosaminyltransferase I. Here we have performed comparative N-glycoproteomic analyses of insertional mutants of mannosidase 1A (IM Man1A ) and xylosyltransferase 1A (IM XylT1A ). The disruption of man1A affected methylation of Man and the addition of terminal Xyl. The absence of XylT1A led to shorter N-glycans compared to the wild type. The use of a IM Man1A xIM XylT1A double mutant revealed that the absence of Man1A suppressed the IM XylT1A phenotype, indicating that the increased N-glycan trimming is regulated by core β1,2-Xyl and is dependent on Man1A activity. These data point toward an enzymatic cascade in the N-glycosylation pathway of C. reinhardtii with interlinked roles of Man1A and XylT1A. The results described herein represent the first step toward a functional characterization of the enzymatic N-glycosylation machinery in C. reinhardtii.

Primary and motile cilia/flagella function as cellular antennae, receiving signals from the environment, and subsequently activating signaling pathways that are critical for cellular homeostasis and differentiation [1-3]. Recent work with the green alga Chlamydomonas and the nematode C. elegans demonstrated that ectosomes can be released from the cilium and can mediate the intercellular communication [4-9]. To better understand the function of flagellar ectosomes, we have compared their protein composition to that of the flagellar membrane from which they are derived. Ectosomes released from flagella have a unique protein composition, being enriched in a subset of flagellar membrane proteins, proteases, proteins from the endosomal sorting complex required for transport (ESCRT) [10-12], small GTPases, and ubiquitinated proteins. Live imaging showed that an ESCRT-related protein (PDCD6) was enriched in ectosomes released from flagella during gamete activation. We devised a sensitive and rapid assay to monitor ectosome release using luciferase fused to PDCD6 and a mutated ubiquitin. Ectosome release increased when cells underwent flagellar resorption. Knockdown of two ESCRT-related proteins, PDCD6 and VPS4, attenuated ectosome release during flagellar shortening and shortening was slowed. These data suggest that the ESCRT proteins mediate ectosome release and thereby influence flagellar shortening in Chlamydomonas. In addition, the prevalence of receptors such as agglutinin and ubiquitinated proteins in ciliary ectosomes suggests that they are involved in cell signaling and turnover of ciliary proteins.

As a well-characterized eukaryotic DNA binding transcription factor, Gal4 has been extensively employed to construct controllable gene expression systems in Saccharomyces cerevisiae. The problem of insufficient inducibility arises in the constructs with multiples genes under control of GAL promoters due to the low expression level and activity of the native Gal4. In order to obtain improved transcriptional regulation systems for multi-gene pathways, Gal4 mutants with improved activity were created by directed evolution. During the preliminary screening, five positive Gal4 variants were isolated based on the lycopene-indicated high-throughput screening method. Analysis of the mutation sites revealed that the majority of positive mutations are localized in the middle homology region with unspecified function, suggesting an important role of this domain in transactivation of Gal4. Through combinatorial site-directed mutagenesis, the best variant Gal4T406A/V413A was obtained, which successfully increased the transcription of PGAL-driven lycopene pathway genes and led to 48 % higher lycopene accumulation relative to the wild-type Gal4. This study demonstrates the viability of modifying Gal4 activity by directed evolution for elevated expression of PGAL-driven genes and therefore enhanced production of the target metabolite.

With special fragrance and various biological properties, linalool has wide applications in food, pharmaceuticals and cosmetics industries. Microbial biosynthesis has become a promising approach to linalool production, however its efficiency is limited by the limited precursor supply and poor efficiency of linalool synthase. With the goal of enhancing heterologous linalool production in Saccharomyces cerevisiae, protein engineering and metabolic engineering were simultaneously employed to solve these bottlenecks. Linalool-producing yeast was first constructed by introduction of t67OMcLIS selected out from a collection of linalool synthases. The efficiency of linalool biosynthesis was then improved by strengthening the supply of geranyl pyrophosphate (GPP) as precursor via overexpression of the complete mevalonate (MVA) pathway and a GPP synthase variant. To accelerate the conversion of the accumulated GPP to linalool, t67OMcLIS was engineered by means of directed evolution after development of a competition-based color-indicated high-throughput screening method. Expression of the t67OMcLIS variant in the engineered S. cerevisiae with enhanced precursor supply yielded 53.14 mg/L of linalool in biphasic shake-flask culture. This study delivered an efficient linalool-producing yeast cell factory and meanwhile provided efficient strategies for biosynthesis of other valuable monoterpenes.

We report a rare case of culture negative L4-L5 discitis and epidural abscess in an immunocompetent child who had dry cupping therapy performed to treat low back strain. The causative pathogen was identified as Stenotrophomonas maltophilia by shotgun metagenomic sequencing of spinal cord aspirate after more than one month of unsuccessful empirical treatment with 6 different antibiotics. The patient was successfully treated with Sulfamethoxazole-trimethoprim and minocycline. Cupping therapy is a very popular medical procedure widely used in China, but the potential risk for severe infections such as discitis and epidural abscess described in this case should be recognized. Keywords: Stenotrophomonas maltophilia, Discitis, Epidural abscess, Shotgun metagenomic sequencing, Dry cupping therapy

The present study aimed to determine the in vitro activities of sulbactam and sitafloxacin against extensively-drug resistant Acinetobacter baumannii (XDR-A. baumannii). A total of 50 strains of XDR-A. baumannii were isolated from clinical specimens. Broth microdilution assay was applied to determine the minimum inhibitory concentration (MIC) for sulbactam and sitafloxacin. Microdilution checkerboard method was used to determine the in vitro activity of this antimicrobial combination. Accordingly, the fractional inhibitory concentration (FIC) and FIC index (FICI) were calculated. Time-kill study was also carried out for four strains with different susceptibilities to determine the bactericidal activities of individual or combined use of sitafloxacin and sulbactam. Isolates with MICs of sitafloxacin ≤2 mg/l were considered to be susceptible to sitafloxacin. The susceptibility rate for sitafloxacin was 92% originally. When combined with sulbactam, this rate increased to 96%. Microdilution checkerboard results indicated that, when tested in combination, sulbactam/sitafloxacin exhibited marked synergistic and partial synergistic effects on 16 and 50% of the 50 strains, respectively. Time-kill assay suggested that sulbactam enhanced the bactericidal activity of sitafloxacin and the combination induced a synergistic effect. For strains that were not susceptible to sitafloxacin, the bactericidal activities of the combination of sitafloxacin and sulbactam at a sub-MIC concentration were impaired. However, this impairment could be overcome with the increase of the concentration to 1X MIC. The present study demonstrated that sulbactam enhanced the in vitro antimicrobial activity of sitafloxacin against XDR-A. baumannii.

Background The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. Results By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained ~150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also constructed, which increased the efficiency of gene interruption. Conclusion In summary, this library provides a multifunctional platform both for obtaining mutants of interested genes and for screening of mutants with specific phenotype.

In the process of bacteriophage and bacteria struggle, adsorption is the key factor to determine who is the winner. In this paper, the molecular mechanism of tailed bacteriophage recognition and adsorption to host and the strategy of "fighting wisdom and courage" between them are reviewed.

We investigated the combined effects of soil moisture and light intensity on the growth, development and ecophysiological characteristics of one-year old Amorpha fruticosa seedlings. Soil moisture and light intensity influenced the ecophysiological characteristics of Amorpha fruticosa seedlings. Soil moisture resulted in the decreases of growth rate, individual size, net photosynthetic rate, transpiration rate, leaf water loss rate (WLR), and biomass accumulation of plant parts, and led to increased leaf water saturation deficit (WSD). Under water stress, more photosynthetic products were allocated to root growth. With decreasing light intensity, net photosynthetic rate, transpiration rate, chla/b, water saturation deficit, water use efficiency, water loss rate and biomass accumulation declined, while Chla, Chlb, Chla+b and carotenoids (Car) increased and more photosynthetic products were allocated to stem and leaf growth. Maximum growth vigor, net photosynthetic rate and total biomass accumulation in Amorpha fruticosa seedlings was recorded at 75–80% soil water-holding capacity and 100% light density in greenhouse environments.

This study investigated the effects of berberine on amelioration of hyperglycemia and hyperlipidemia and the mechanism involved in high glucose and high fat diet-induced diabetic hamsters. Golden hamsters fed with high glucose and high fat diet were medicated with metformin, simvastatin, and low or high dose of berberine (50 and 100 mg·kg−1) for 6 weeks. The results showed that the body weights were significantly lower in berberine-treated groups than control group. Histological analyses revealed that the treatment of berberine inhibited hepatic fat accumulation. Berberine significantly reduced plasma total cholesterol, triglyceride, free fatty acid, low density lipoprotein cholesterol, malondialdehyde, thiobarbituric acid-reactive substance, and 8-isoprostane level but significantly increased plasma superoxide dismutase activity. Glucose and insulin levels were significantly reduced in metformin and berberine-treated groups. Glucose tolerance tests documented that berberine-treated mice were more glucose tolerant. Berberine treatment increased expression of skeletal muscle glucose transporter 4 mRNA and significantly decreased liver low density lipoprotein receptor mRNA expression. The study suggested that berberine was effective in lowering blood glucose and lipids levels, reducing the body weight, and alleviating the oxidative stress in diabetic hamsters, which might be beneficial in reducing the cardiovascular risk factors in diabetes.