Programmed genome editing by a miniature CRISPR-Cas12f nuclease.

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.

Improvement of protein production by engineering a novel antiapoptotic baculovirus vector to suppress the expression of Sf-caspase-1 and Tn-caspase-1.

The baculovirus expression vector system (BEVS) is an attractive manufacturing platform for recombinant protein production in insect cells. However, baculovirus infection commonly induces host apoptosis in 3-4 days which would subsequently terminate the protein expression. Previous studies have proved that protein production by BEVS can be elevated in apoptosis-suppressed insect cells. We also developed a baculovirus vector in our previous report to inhibit the apoptosis and improve protein production in Sf9 cells. In this study, we designed five short hairpin RNA (shRNA) expression cassettes targeting a conserved region in Spodoptera frugiperda caspase-1 (Sf-caspase-1) and Trichoplusia ni caspase-1 (Tn-caspase-1), and found that introduction of C to T mutations within the stem region of the expression cassette was beneficial for the heterologous protein expression. One of the improved shRNA expression cassettes was knocked into a bacmid with the deletion of several nonessential genes. The novel baculovirus vector demonstrated the ability to suppress cell apoptosis in both Sf9 and High Five cells, and exhibited superior recombinant protein productivity of intracellularly expressed GFP and firefly luciferase and secreted glycoprotein OD-Fc. The antiapoptotic baculovirus vector developed in this study could serve as a useful tool for the protein production in scientific research and pharmaceutical industries.

Chlorogenic acid inhibits proliferation in human hepatoma cells by suppressing noncanonical NF-κB signaling pathway and triggering mitochondrial apoptosis.

Chlorogenic acid (CGA), a phenylpropanoid derived from Eucommia ulmoides Oliver, has been shown to exhibit potent cytotoxic and anti-proliferative activities against several human cancers. However, the effects of CGA on hepatocellular carcinoma (HCC) and the underlying mechanisms have not been intensively studied. In this study, the CGA treatment effects on the viability of human hepatoma cells were investigated by MTT assay. Our data showed that CGA could dose-dependently inhibit the activity of human hepatoma cells Hep-G2 and Huh-7, but did not affect the activity and growth of normal human hepatocyte QSG-7701. The genes and pathways influenced by CGA treatment were explored by RNA sequencing and bioinformatics analysis, which identified 323 differentially expressed genes (DEGs) involved in multiple pharmacological signaling pathways such as MAPK, NF-κB, apoptosis and TGF-ß signaling pathways. Further analyses by real-time quantitative PCR, Western blot and flow cytometry revealed that CGA effectually suppressed the noncanonical NF-κB signaling pathway, meanwhile it activated the mitochondrial apoptosis of HCC by upregulation of the BH3-only protein Bcl-2 binding component 3 (BBC3). Our findings demonstrated the potential of CGA in suppressing human hepatoma cells and provided a new insight into the anti-cancer mechanism of CGA.

Immersion immunization with recombinant baculoviruses displaying cyprinid herpesvirus 2 membrane proteins induced protective immunity in gibel carp.

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp.

Smart Camera Aware Crowd Counting via Multiple Task Fractional Stride Deep Learning.

Estimating the number of people in highly clustered crowd scenes is an extremely challenging task on account of serious occlusion and non-uniformity distribution in one crowd image. Traditional works on crowd counting take advantage of different CNN like networks to regress crowd density map, and further predict the count. In contrast, we investigate a simple but valid deep learning model that concentrates on accurately predicting the density map and simultaneously training a density level classifier to relax parameters of the network to prevent dangerous stampede with a smart camera. First, a combination of atrous and fractional stride convolutional neural network (CAFN) is proposed to deliver larger receptive fields and reduce the loss of details during down-sampling by using dilated kernels. Second, the expanded architecture is offered to not only precisely regress the density map, but also classify the density level of the crowd in the meantime (MTCAFN, multiple tasks CAFN for both regression and classification). Third, experimental results demonstrated on four datasets (Shanghai Tech A (MAE = 88.1) and B (MAE = 18.8), WorldExpo'10(average MAE = 8.2), NS UCF_CC_50(MAE = 303.2) prove our proposed method can deliver effective performance.

Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. IMPORTANCE: It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

Bufospirostenin A and Bufogargarizin C, Steroids with Rearranged Skeletons from the Toad Bufo bufo gargarizans.

Bufospirostenin A (1) and bufogargarizin C (2), two novel steroids with rearranged A/B rings, were isolated from the toad Bufo bufo gargarizans. Compound 1 represents the first spirostanol found in animals. Compound 2 is an unusual bufadienolide with a cycloheptatriene B ring. Their structures were elucidated by spectroscopic analysis, single crystal X-ray diffraction analysis, and computational calculations.

Spectroscopic Study on the Interaction between Naphthalimide-Polyamine Conjugates and Bovine Serum Albumin (BSA).

The effect of a naphthalimide pharmacophore coupled with diverse substituents on the interaction between naphthalimide-polyamine conjugates 1-4 and bovine serum albumin (BSA) was studied by UV absorption, fluorescence and circular dichroism (CD) spectroscopy under physiological conditions (pH = 7.4). The observed spectral quenching of BSA by the compounds indicated that they could bind to BSA. Furthermore, caloric fluorescent tests revealed that the quenching mechanisms of compounds 1-3 were basically static type, but that of compound 4 was closer to a classical type. The Ksv values at room temperature for compound-BSA complexes-1-BSA, 2-BSA, 3-BSA and 4-BSA were 1.438 × 104, 3.190 × 104, 5.700 × 104 and 4.745 × 105, respectively, compared with the value of MINS, 2.863 × 104 at Ex = 280 nm. The obtained quenching constant, binding constant and thermodynamic parameter suggested that the binding between compounds 1-4 with BSA protein, significantly affected by the substituted groups on the naphthalene backbone, was formed by hydrogen bonds, and other principle forces mainly consisting of charged and hydrophobic interactions. Based on results from the analysis of synchronous three-dimensional fluorescence and CD spectra, we can conclude that the interaction between compounds 1-4 and BSA protein has little impact on the BSA conformation. Calculated results obtained from in silico molecular simulation showed that compound 1 did not prefer either enzymatic drug sites I or II over the other. However, DSII in BSA was more beneficial than DSI for the binding between compounds 2-4 and BSA protein. The binding between compounds 1-3 and BSA was hydrophobic in nature, compared with the electrostatic interaction between compound 4 and BSA.

Phase separation in DNA double-strand break response.

DNA double-strand break (DSB) is the most dangerous type of DNA damage, which may lead to cell death or oncogenic mutations. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two typical DSB repair mechanisms. Recently, many studies have revealed that liquid-liquid phase separation (LLPS) plays a pivotal role in DSB repair and response. Through LLPS, the crucial biomolecules are quickly recruited to damaged sites with a high concentration to ensure DNA repair is conducted quickly and efficiently, which facilitates DSB repair factors activating downstream proteins or transmitting signals. In addition, the dysregulation of the DSB repair factor's phase separation has been reported to promote the development of a variety of diseases. This review not only provides a comprehensive overview of the emerging roles of LLPS in the repair of DSB but also sheds light on the regulatory patterns of phase separation in relation to the DNA damage response (DDR).

Characteristics of the gut microbiota and serum metabolites in postmenopausal women with reduced bone mineral density.

Introduction: Emerging evidence suggests that the gut microbiota is closely associated with bone homeostasis. However, little is known about the relationships among the bone mineral density (BMD) index, bone turnover markers, and the gut microbiota and its metabolites in postmenopausal women. Methods: In this study, to understand gut microbiota signatures and serum metabolite changes in postmenopausal women with reduced BMD, postmenopausal individuals with normal or reduced BMD were recruited and divided into normal and OS groups. Feces and serum samples were collected for 16S rRNA gene sequencing, liquid chromatography coupled with mass spectrometry (LC-MS)-based metabolomics and integrated analysis. Results: The results demonstrated that bacterial richness and diversity were greater in the OS group than in the normal group. Additionally, distinguishing bacteria were found among the two groups and were closely associated with the BMD index and bone turnover markers. Metabolomic analysis revealed that the expression of serum metabolites, such as etiocholanolone, testosterone sulfate, and indole-3-pyruvic acid, and the corresponding signaling pathways, especially those involved in tryptophan metabolism, fatty acid degradation and steroid hormone biosynthesis, also changed significantly. Correlation analysis revealed positive associations between normal group-enriched Bacteroides abundance and normal group-enriched etiocholanolone and testosterone sulfate abundances; in particular, Bacteroides correlated positively with BMD. Importantly, the tryptophan-indole metabolism pathway was uniquely metabolized by the gut bacteria-derived tnaA gene, the predicted abundance of which was significantly greater in the normal group than in the control group, and the abundance of Bacteroides was strongly correlated with the tnaA gene. Discussion: Our results indicated a clear difference in the gut microbiota and serum metabolites of postmenopausal women. Specifically altered bacteria and derived metabolites were closely associated with the BMD index and bone turnover markers, indicating the potential of the gut microbiota and serum metabolites as modifiable factors and therapeutic targets for preventing osteoporosis.

Resting-State Network Analysis Reveals Altered Functional Brain Connectivity in Essential Tremor.

INTRODUCTION: Essential tremor (ET) comprises motor and non-motor related features, while the current neuro-pathogenetic basis is still insufficient to explain the etiologies of ET. While cerebellum associated circuits have been discovered, the large-scale cerebral network connectivity in ET remains unclear. This study aimed to characterize the ET in terms of functional connectivity as well as network. We hypothesized that the resting-state network within cerebrum could be altered in ET patients. METHODS: Resting-state functional MRI (fMRI) was used to evaluate the inter- and intra-network connectivity as well as the functional activity in ET and normal control. Correlation analysis was performed to explore the relationship between resting-state network metrics and tremor features. RESULTS: Comparison of inter-network connectivity indicated a decreased connectivity between default mode network and ventral attention network in ET group (P<0.05). Differences in functional activity (assessed by amplitude of low frequency fluctuation, ALFF) were found in several brain regions participating in various resting-state networks (P<0.05). ET group generally have higher degree centrality over normal control. Correlation analysis has revealed that tremor features are associated with inter-network connectivity (|r|=0.135-0.506), ALFF (|r|=0.313-0.766), and degree centrality (|r|=0.523-0.710). CONCLUSION: Alterations in the cerebral network of ET was detected by using resting-state fMRI, demonstrating a potentially useful approach to explore the cerebral alterations in ET.

Detection of myeloma cell-derived microvesicles: a tool to monitor multiple myeloma load.

The persistence of tumor load in multiple myeloma (MM) lead to relapse in patients achieving complete remission (CR). Appropriate and effective methods of myeloma tumor load monitoring are important for guiding clinical management. This study aimed to clarify the value of microvesicles in monitoring MM tumor load. Microvesicles in bone marrow and peripheral blood were isolated by differential ultracentrifugation and detected by flow cytometry. Western blotting was applied to assess myosin light chain phosphorylation levels. Flow cytometry to detect Ps+CD41a-, Ps+CD41a-CD138+, Ps+CD41a-BCMA+ microvesicles from bone marrow can be used to predict myeloma burden, furthermore, Ps+CD41a- microvesicles may as a potential index to MRD test. Mechanistically, the releasing of microvesicles from MM cell was regulated by Pim-2 Kinase via Phosphorylation of MLC-2 protein.

Effect of low temperature on the shaping of yeast-derived metabolite compositions during wine fermentation.

Low-temperature fermentation is considered to enrich the aroma of wine. The metabolism of Saccharomyces cerevisiae responding to low temperatures is intricate and this complexity is further enhanced by various strains and culture media. However, the real effects of low-temperature fermentation on yeast metabolism are unclear. Aiming to clarify the yeast-derived metabolite formation in a low-temperature winemaking range, fermentations were performed at 10, 15, and 20 °C, using five wine yeast strains in two media respectively. Tolerance toward low temperatures and metabolite compositions (including basic chemical compositions and volatile aroma compounds) of wine yeasts were analyzed. Results showed that ethanol, ethyl acetate, and ethyl butanoate increased with the temperature decreasing, while acetic acid, phenylethanol, phenylethyl acetate, ethyl decanoate, and ethyl hexadecanoate decreased with decreasing temperature. The linear relationship between fermentation temperature and the formation of ethanol, acetic acid, and phenylethanol might be fundamentally due to the growth changes caused by temperature. The enhanced production of ethyl acetate and ethyl butyrate followed by decreasing temperature probably resulted from low-temperature-stimulated enzymes in metabolic pathways. These findings reveal a typical profile of yeast-derived metabolites at low-temperature fermentation and provide evidence to support the application of low-temperature winemaking in the wine industry.

ß-Cyanoalanine Synthase Regulates the Accumulation of ß-ODAP via Interaction with Serine Acetyltransferase in Lathyrus sativus.

ß-N-Oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), found in Lathyrus sativus at first, causes a neurological disease, lathyrism, when over ingested in an unbalanced diet. Our previous research suggested that ß-ODAP biosynthesis is related to sulfur metabolism. In this study, ß-cyanoalanine synthase (ß-CAS) was confirmed to be responsible for ß-ODAP biosynthesis via in vitro enzymatic analysis. LsCAS was found to be pyridoxal phosphate (PLP)-dependent via spectroscopic analysis and dual functional via enzymatic activity analysis. Generation of a M135T/M235S/S239T triple mutant of LsCAS, which are the key sites to control the ratio of CAS/cysteine synthase (CS) activity, switches reaction chemistry to that of a CS. LsCAS interactions were further screened and verified via Y2H, BiFC and pull-down assay. It was suggested that LsSAT2 interacts and forms a cysteine regulatory complex (CRC) with LsCAS in mitochondria, which improves LsSAT while reduces LsCAS activities to affect ß-ODAP content positively. These results provide new insights into the molecular regulation of ß-ODAP content in L. sativus.

Non-Invasive Remote Temperature Monitoring Using Microwave-Induced Thermoacoustic Imaging.

Non-invasive temperature monitoring of tissue at depth in real-time is critical to hyperthermia therapies such as high-intensity focused ultrasound. Knowledge of temperature allows for monitoring treatment as well as providing real-time feedback to adjust deposited power in order to maintain safe and effective temperatures. Microwave-induced thermoacoustic (TA) imaging, which combines the conductivity/dielectric contrast of microwave imaging with the resolution of ultrasound, shows potential for estimating temperature non-invasively in real-time by indirectly measuring the temperature dependent parameters from reconstructed images. In this work, we study the temperature dependent behavior of the generated pressure in the TA effect and experimentally demonstrate simultaneous imaging and temperature monitoring using TA imaging. The proof-of-concept experiments demonstrate millimeter spatial resolution while achieving degree-level accuracy.

Coordinated Regulation of Grape Berry Flesh Color by Transcriptional Activators and Repressors.

Yan73 is a teinturier Vitis vinifera variety with red berry flesh, but the molecular mechanisms underlying its flesh coloration remain unclear. We analyzed the flavonoid metabolic and transcriptome profiles of Yan73 berry red and white flesh using HPLC-ESI-MS/MS and RNA-sequencing technologies. Anthocyanins are the main flavonoids responsible for Yan73 berry flesh color, and the coloration is coordinately regulated by the VvMYBA1 transcriptional activator and VvMYBC2-L1 transcriptional repressor. Furthermore, yeast one- and two-hybrid, dual luciferase, and bimolecular fluorescence complementation assays suggested that VvMYBA1 positively regulates Yan73 berry flesh color via interactions with VvWDR1 and the activation of the VvCHI3, VvOMT, and VvGST4 promoters, whereas VvMYBC2-L1 negatively regulates Yan73 berry flesh color, possibly by competing with the R2R3-MYB transcriptional activators for bHLH partners or by repressing VvOMT and VvGST4 expression. Our findings provide new insights into the molecular mechanisms regulating grape flesh color.

A viral expression factor behaves as a prion.

Prions are proteins that can fold into multiple conformations some of which are self-propagating. Such prion-forming proteins have been found in animal, plant, fungal and bacterial species, but have not yet been identified in viruses. Here we report that LEF-10, a baculovirus-encoded protein, behaves as a prion. Full-length LEF-10 or its candidate prion-forming domain (cPrD) can functionally replace the PrD of Sup35, a widely studied prion-forming protein from yeast, displaying a [PSI+]-like phenotype. Furthermore, we observe that high multiplicity of infection can induce the conversion of LEF-10 into an aggregated state in virus-infected cells, resulting in the inhibition of viral late gene expression. Our findings extend the knowledge of current prion proteins from cellular organisms to non-cellular life forms and provide evidence to support the hypothesis that prion-forming proteins are a widespread phenomenon in nature.

Metabolic acidosis in critically ill patients with cirrhosis: Epidemiology and short-term mortality risk factors.

BACKGROUND/AIMS: Metabolic acidosis is a common complication in patients with cirrhosis at the intensive care units (ICUs) and associated with increased mortality. The aim of our research was to explore the epidemiology and risk factors of metabolic acidosis in critically ill patients with cirrhosis. MATERIALS AND METHODS: A total of 975 patients with cirrhosis were selected into our study, and all participants were followed up for at least 28 days. Cox regression model and machine-learning algorithm were used to identify the importance of different risk factors, respectively. Finally, an improved prognostic model as Model for End-stage Liver Disease and metabolic acidosis (MELD-MA) was developed. RESULTS: Among the 975 patients with liver cirrhosis, 506 had metabolic acidosis, including 257 patients who had decompensated metabolic acidosis at ICU admission. The 28-day mortality was 41% (206/506) in patients with metabolic acidosis. Bilirubin (hazard ratio (HR): 1.023, 95% confidence interval (CI): 1.011-1.036), international normalized ratio (HR: 1.527, 95% CI: 1.332-1.750), pH (HR: 0.173, 95% CI: 0.047-0.640), BE-Lac (HR: 0.907, 95% CI: 0.868-0.948), and BE-Na (HR: 0.923, 95% CI: 0.859-0.991) were considered as independent prognostic parameters for 28-day mortality. MELD-NA had significantly higher discrimination (area under the receiver operating characteristic curve 0.79) than MELD and Child-Pugh score. CONCLUSION: Critically ill patients with cirrhosis have a high mortality rate and poor prognosis because of the high prevalence of metabolic acidosis. Lactic acidosis is the worst prognosis of all types of metabolic acidosis. MELD-MA performs well on the short-term mortality assessment in critically ill patients with cirrhosis and metabolic acidosis.

The Network of Interactions Among Porcine Reproductive and Respiratory Syndrome Virus Non-structural Proteins.

The RNA synthesis of porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus, is compartmentalized in virus-induced double-membrane vesicles where viral proteins and some cellular proteins assemble into replication and transcription complexes (RTCs). The viral replicase proteins are the major components of the RTCs but the physical associations among these non-structural proteins (nsps) remain elusive. In this study, we investigated the potential interactions between PRRSV nsps by yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and pull-down assays. Our analyses revealed a complex network of interactions involving most of PRRSV nsps. Among them, nsp9 and nsp12 were identified as the hubs of the nsp interactome; transmembrane proteins nsp2 and nsp5 both interacted with nsp3, indicating that the three membrane-bound proteins might bind together to form the scaffold to support the association of RTCs with the intracellular membrane. The PRRSV nsp interactions identified in this study may provide valuable clues for future researches on the RTC formation and function.