Creating a functional single-chromosome yeast.

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.

Nomogram for predicting survival in patients with advanced hepatocellular carcinoma treated with PD-1 inhibitors: incorporating pre-treatment and post-treatment clinical parameters.

BACKGROUND: Immunotherapy has transformed cancer treatment patterns for advanced hepatocellular carcinoma (aHCC) in recent years. Therefore, the identification of predictive biomarkers has important clinical implications. METHODS: We collected medical records from 117 aHCC patients treated with anti-PD-1 antibody. Kaplan-Meier analysis and Cox proportional hazard regression were used to evaluate the association between peripheral blood biomarkers and overall survival (OS) and progression-free survival (PFS). Finally, the prognostic nomogram was constructed. RESULTS: The mPFS and mOS were 7.0 months and 18.7 months, respectively. According to Kaplan-Meier analysis and Cox regression analysis, we regarded the treatment regimen (p = 0.020), hemoglobin (Hb) at 6-week (p = 0.042), neutrophil-to-lymphocyte ratio (NLR) at 6-week (p < 0.001), system immune inflammation index (SII) at 6-week (p = 0.125) as predictors of PFS, and alpha fetoprotein (AFP) (p = 0.035), platelet-to-lymphocyte ratio (PLR) (p = 0.012), Hb at 6-week (p = 0.010) and NLR at 6-week (p = 0.020) as predictors of OS. Furthermore, the results suggest that the OS and PFS nomogram model were in agreement with actual observations. CONCLUSION: Biomarkers in peripheral blood can predict the prognosis of patients with aHCC treated with anti-PD-1 antibody. The development of nomogram models can help us to screen potential patients who can benefit from immunotherapy.

The Anti-Allergic Rhinitis Effect of Traditional Chinese Medicine of Shenqi by Regulating Mast Cell Degranulation and Th1/Th2 Cytokine Balance.

Shenqi is a traditional Chinese polyherbal medicine has been widely used for the treatment of allergic rhinitis (AR). The aim of this study was to investigate the anti-allergic rhinitis activity of Shenqi and explore its underlying molecular mechanism. Ovalbumin (OVA)-induced allergic rhinitis rat model was used to evaluate the anti-allergic rhinitis effect of Shenqi. The effect of Shenqi on IgE-mediated degranulation was measured using rat basophilic leukemia (RBL-2H3) cells. Primary spleen lymphocytes were isolated to investigate the anti-allergic mechanism of Shenqi by detecting the expression of transcription factors via Western blot and the level of cytokines (IL-4 and IFN-γ) via ELISA. In OVA-induced AR rat models, Shenqi relieved the allergic rhinitis symptoms, inhibited the histopathological changes of nasal mucosa, and reduced the levels of IL-4 and IgE. The results from the in vitro study certified that Shenqi inhibited mast cell degranulation. Furthermore, the results of GATA3, T-bet, p-STAT6, and SOCS1 expression and production of IFN-γ and IL-4 demonstrated that Shenqi balanced the ratio of Th1/Th2 (IFN-γ/IL-4) in OVA-stimulated spleen lymphocytes. In conclusion, these results suggest that Shenqi exhibits an obvious anti-allergic effect by suppressing the mast cell-mediated allergic response and by improving the imbalance of Th1/Th2 ratio in allergic rhinitis.

Wilfordonols A-D: four new norsesquiterpenes from the leaves of Tripterygium wilfordii.

Four new norsesquiterpenes wilfordonols A-D (1-4), along with three known compounds, sarmentol B (5), boscialin (6), and (+)-loliolide (7), were isolated from the leaves of Tripterygium wilfordii Hook.f.. The structures of the new compounds were elucidated on the basis of their spectroscopic analysis, and the absolute configuration of the compounds was confirmed by CD and modified Mosher's method. At a concentration of 10 µM, compounds 4, 6, and 7 inhibited signal transducer and activator of transcription 1 translocation by 34.27 ± 1.02%, 48.93 ± 1.76%, and 70.31 ± 2.20%, respectively.

Elimination of subtelomeric repeat sequences exerts little effect on telomere essential functions in Saccharomyces cerevisiae.

Telomeres, which are chromosomal end structures, play a crucial role in maintaining genome stability and integrity in eukaryotes. In the baker's yeast Saccharomyces cerevisiae, the X- and Y'-elements are subtelomeric repetitive sequences found in all 32 and 17 telomeres, respectively. While the Y'-elements serve as a backup for telomere functions in cells lacking telomerase, the function of the X-elements remains unclear. This study utilized the S. cerevisiae strain SY12, which has three chromosomes and six telomeres, to investigate the role of X-elements (as well as Y'-elements) in telomere maintenance. Deletion of Y'-elements (SY12YΔ), X-elements (SY12XYΔ+Y), or both X- and Y'-elements (SY12XYΔ) did not impact the length of the terminal TG1-3 tracks or telomere silencing. However, inactivation of telomerase in SY12YΔ, SY12XYΔ+Y, and SY12XYΔ cells resulted in cellular senescence and the generation of survivors. These survivors either maintained their telomeres through homologous recombination-dependent TG1-3 track elongation or underwent microhomology-mediated intra-chromosomal end-to-end joining. Our findings indicate the non-essential role of subtelomeric X- and Y'-elements in telomere regulation in both telomerase-proficient and telomerase-null cells and suggest that these elements may represent remnants of S. cerevisiae genome evolution. Furthermore, strains with fewer or no subtelomeric elements exhibit more concise telomere structures and offer potential models for future studies in telomere biology.

PVR-AFM: A Pathological Voice Repair System based on Non-linear Structure.

OBJECTIVE: Speech signal processing has become an important technique to ensure that the voice interaction system communicates accurately with the user by improving the clarity or intelligibility of speech signals. However, most existing works only focus on whether to process the voice of average human but ignore the communication needs of individuals suffering from voice disorder, including voice-related professionals, older people, and smokers. To solve this demand, it is essential to design a non-invasive repair system that processes pathological voices. METHODS: In this paper, we propose a repair system for multiple polyp vowels, such as /a/, /i/ and /u/. We utilize a non-linear model based on amplitude-modulation (AM) and a frequency-modulation (FM) structure to extract the pitch and formant of pathological voice. To solve the fracture and instability of pitch, we provide a pitch extraction algorithm, which ensures that pitch's stability and avoids the errors of double pitch caused by the instability of low-frequency signal. Furthermore, we design a formant reconstruction mechanism, which can effectively determine the frequency and bandwidth to accomplish formant repair. RESULTS: Finally, spectrum observation and objective indicators show that the system has better performance in improving the intelligibility of pathological speech.

Construction and Evaluation of Recombinant Pseudorabies Virus Expressing African Swine Fever Virus Antigen Genes.

African swine fever (ASF) is a highly contact infectious disease caused by the African swine fever virus (ASFV). The extremely complex structure and infection mechanism make it difficult to control the spread of ASFV and develop the vaccine. The ASFV genome is huge with many antigenic genes. Among them, CP204L (p30), CP530R (pp62), E183L (p54), B646L (p72), and EP402R (CD2v) are involved in the process of the virus cycle, with strong immunogenicity and the ability to induce the body to produce neutralizing antibodies. In this study, the recombinant virus rBartha-K61-pASFV that expresses the above ASFV antigen genes was constructed by Red/ET recombineering technology using pseudorabies virus (PRV) vaccine strain Bartha-K61. Western blot analysis showed that the ASFV antigen gene was expressed and the recombinant virus showed good genetic stability and proliferation characteristics in 15 continuous generations on porcine kidney (PK15) cells. The results of immunoassay of piglets and mice showed that rBartha-K61-pASFV had good immunogenicity and could induce higher antibody levels in the body. Therefore, PRV was a promising viral vector for expressing the ASFV antigen gene, and all the experiments in this study laid a foundation for the further development of a new viral vector vaccine of ASFV.

Multiple Vowels Repair Based on Pitch Extraction and Line Spectrum Pair Feature for Voice Disorder.

Individuals, such as voice-related professionals, elderly people and smokers, are increasingly suffering from voice disorder, which implies the importance of pathological voice repair. Previous work on pathological voice repair only concerned about sustained vowel /a/, but multiple vowels repair is still challenging due to the unstable extraction of pitch and the unsatisfactory reconstruction of formant. In this paper, a multiple vowels repair based on pitch extraction and Line Spectrum Pair feature for voice disorder is proposed, which broadened the research subjects of voice repair from only single vowel /a/ to multiple vowels /a/, /i/ and /u/ and achieved the repair of these vowels successfully. Considering deep neural network as a classifier, a voice recognition is performed to classify the normal and pathological voices. Wavelet Transform and Hilbert-Huang Transform are applied for pitch extraction. Based on Line Spectrum Pair (LSP) feature, the formant is reconstructed. The final repaired voice is obtained by synthesizing the pitch and the formant. The proposed method is validated on Saarbrücken Voice Database (SVD) database. The achieved improvements of three metrics, Segmental Signal-to-Noise Ratio, LSP distance measure and Mel cepstral distance measure, are respectively 45.87%, 50.37% and 15.56%. Besides, an intuitive analysis based on spectrogram has been done and a prominent repair effect has been achieved.

Cdc13 is predominant over Stn1 and Ten1 in preventing chromosome end fusions.

Telomeres define the natural ends of eukaryotic chromosomes and are crucial for chromosomal stability. The budding yeast Cdc13, Stn1 and Ten1 proteins form a heterotrimeric complex, and the inactivation of any of its subunits leads to a uniformly lethal phenotype due to telomere deprotection. Although Cdc13, Stn1 and Ten1 seem to belong to an epistasis group, it remains unclear whether they function differently in telomere protection. Here, we employed the single-linear-chromosome yeast SY14, and surprisingly found that the deletion of CDC13 leads to telomere erosion and intrachromosome end-to-end fusion, which depends on Rad52 but not Yku. Interestingly, the emergence frequency of survivors in the SY14 cdc13Δ mutant was ~29 fold higher than that in either the stn1Δ or ten1Δ mutant, demonstrating a predominant role of Cdc13 in inhibiting telomere fusion. Chromosomal fusion readily occurred in the telomerase-null SY14 strain, further verifying the default role of intact telomeres in inhibiting chromosome fusion.

Creating functional chromosome fusions in yeast with CRISPR-Cas9.

CRISPR-Cas9-facilitated functional chromosome fusion allows the generation of a series of yeast strains with progressively reduced chromosome numbers that are valuable resources for the study of fundamental concepts in chromosome biology, including replication, recombination and segregation. We created a new yeast strain with a single chromosome by using the protocol for chromosome fusion described herein. To ensure the accuracy of chromosome fusions in yeast, the long redundant repetitive sequences near linear chromosomal ends are deleted, and the fusion orders are correspondingly determined. Possible influence on gene expression is minimized to retain gene functionality. This protocol provides experimentally derived guidelines for the generation of functional chromosome fusions in yeast, especially for the deletion of repetitive sequences, the determination of the fusion order and cleavage sites, and primary evaluation of the functionality of chromosome fusions. Beginning with design, one round of typical chromosome fusion and functional verifications can be accomplished within 18 d.

Commercial vaccine against pseudorabies virus: A hidden health risk for dogs.

Pseudorabies virus (PRV) is considered as an infectious agent with a wide of host range, causing considerable economic losses in animal husbandry. Although the commercial vaccine against PRV plays an critical role in control of this disease in swine industry, the potential risk of commercial vaccines against PRV for other host is unclear. Here, we report that the commercial vaccine against PRV is a hidden health risk for dogs. We found that different attenuated PRV strains in commercial vaccines possess different tissue tropism, and that the attenuated PRV strains are lethal to dogs, and that the attenuated PRV strain possesses the ability to spread horizontally among the dogs. Collectively, our findings provide clues that the commercial vaccine against PRV is a hidden risk for dogs, even for the owner of pet dogs to take seriously.

CRISPR-Cas9 Facilitated Multiple-Chromosome Fusion in Saccharomyces cerevisiae.

Eukaryotic cells usually contain multiple linear chromosomes. Recently, we artificially created a functional single-chromosome yeast via sequential two-chromosome fusion utilizing the high performance of the CRISPR-Cas9 system and homologous recombination in Saccharomyces cerevisiae. In this paper, we adapted this method for the simultaneous fusion of multiple chromosomes. We demonstrated the fusion of two, two-chromosome sets with a 75% positive rate and three-chromosome fusions with a 50% positive rate. We also found that by using an additional selection marker, the positive rate of two-chromosome fusions reached 100%. Due to the simplicity, efficiency, and portability of this method, we expect that it can be easily adapted for multiple-chromosome fusions in other organisms.

MEGA (Multiple Essential Genes Assembling) deletion and replacement method for genome reduction in Escherichia coli.

Top-down reduction of the bacterial genome to construct desired chassis cells is important for synthetic biology. However, the current progress in the field of genome reduction is greatly hindered by indispensable life-essential genes that are interspersed throughout the chromosomal loci. Here, we described a new method designated as "MEGA (Multiple Essential Genes Assembling) deletion and replacement" that functions by assembling multiple essential genes in an E. coli-S. cerevisiae shuttle vector, removing targeted chromosomal regions containing essential and nonessential genes using a one-round deletion, and then integrating the cloned essential genes into the in situ chromosomal loci via I-SceI endonuclease cleavage. As a proof of concept, we separately generated three large deletions (80-205 kbp) in the E. coli MDS42 chromosome. We believe that the MEGA deletion and replacement method has potential to become widely used in large-scale genome reductions in other sequenced organisms in addition to E. coli.