Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Metastasis.

Most cancer-associated deaths occur due to metastasis, yet our understanding of metastasis as an evolving, heterogeneous, systemic disease and of how to effectively treat it is still emerging. Metastasis requires the acquisition of a succession of traits to disseminate, variably enter and exit dormancy, and colonize distant organs. The success of these events is driven by clonal selection, the potential of metastatic cells to dynamically transition into distinct states, and their ability to co-opt the immune environment. Here, we review the main principles of metastasis and highlight emerging opportunities to develop more effective therapies for metastatic cancer.

Nasal Bacterial Microbiome Differs Between Healthy Controls and Those With Asthma and Allergic Rhinitis.

Perturbation of the microbiome has numerous associations with the phenotypes and progression in chronic airways disease. However, the differences in the nasal microbiome in asthma and allergic rhinitis (AR) have not been defined. We examined whether the nasal microbiome would vary among different comorbidities in asthma and AR and that those differences may be associated with the severity of asthma. Nasal lavage fluid was collected from 110 participants, including 20 healthy controls, 30 subjects with AR, 30 subjects with asthma and 30 subjects with combined asthma + AR. The Asthma Control Questionnaire (ACQ-7) was used to evaluate asthma control status. Using 16S rRNA bacterial gene sequencing, we analyzed nasal microbiome in patients with asthma, AR, combined asthma + AR, and healthy controls. Bacterial diversity was analyzed in corresponding with α diversity indices (Chao and Shannon index). Compared with healthy controls, the Chao index tended to be lower in subjects with AR (P = 0.001), asthma (P = 0.001), and combined asthma + AR (P = 0.001) when compared with healthy controls. Furthermore, the Shannon index was significantly lower in subjects with asthma (P = 0.013) and comorbid asthma with AR (P = 0.004) than the control subjects. Disparity in the structure and composition of nasal bacteria were also observed among the four groups. Furthermore, patients with combined asthma + AR and isolated asthma were divided into two groups according to the level of disease control: partially or well-controlled and uncontrolled asthma. The mean relative abundance observed in the groups mentioned the genera of Pseudoflavonifractor were dominated in patients with well and partially controlled disease, in both isolated asthma and combined asthma + AR. In subjects with uncontrolled asthma and combined asthma + AR, a lower evenness and richness (Shannon index, P = 0.040) was observed in nasal microbiome composition. Importantly, lower evenness and richness in the nasal microbiome may be associated with poor disease control in combined asthma + AR. This study showed the upper airway microbiome is associated with airway inflammation disorders and the level of asthma control.

Clinical features and predictors for patients with severe SARS-CoV-2 pneumonia at the start of the pandemic: a retrospective multicenter cohort study.

BACKGROUND: This study was performed to investigate clinical features of patients with severe SARS-CoV-2 pneumonia and identify risk factors for converting to severe cases in those who had mild to moderate diseases at the start of the pandemic in China. METHODS: In this retrospective, multicenter cohort study, patients with mild to moderate SARS-CoV-2 pneumonia were included. Demographic data, symptoms, laboratory values, and clinical outcomes were collected. Data were compared between non-severe and severe patients. RESULTS: 58 patients were included in the final analysis. Compared with non-severe cases, severe patients with SARS-CoV-2 pneumonia had a longer: time to clinical recovery (12·9 ± 4·4 vs 8·3 ± 4·7; P = 0·0011), duration of viral shedding (15·7 ± 6·7 vs 11·8 ± 5·0; P = 0·0183), and hospital stay (20·7 ± 1·2 vs 14·4 ± 4·3; P = 0·0211). Multivariate logistic regression indicated that lymphocyte count was significantly associated with the rate of converting to severe cases (odds ratio 1·28, 95%CI 1·06-1·54, per 0·1 ×  109/L reduced; P = 0·007), while using of low-to-moderate doses of systematic corticosteroids was associated with reduced likelihood of converting to a severe case (odds ratio 0·14, 95%CI 0·02-0·80; P = 0·0275). CONCLUSIONS: The low peripheral blood lymphocyte count was an independent risk factor for SARS-CoV-2 pneumonia patients converting to severe cases. However, this study was carried out right after the start of the pandemic with small sample size. Further prospective studies are warranted to confirm these findings. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000029839 . Registered 15 February 2020 - Retrospectively registered.

Diagnostic role of liquid-based cytology of bronchial lavage fluid in addition to bronchial brushing specimens in lung cancer.

BACKGROUND: Liquid-based cytology (LBC) tests, including the liquid-based thin layer method, have demonstrated the highest potential for reducing false-negatives and improving sample quality. METHOD: This study aimed to evaluate the diagnostic role of LBC of bronchial brushing specimens in lung cancer. A total of 249 patients were analyzed in our study, involving 155 patients with combined bronchial brushing and bronchoalveolar lavage (BAL) and 94 patients with BAL alone. RESULTS: The sensitivity in the combined bronchial brushing and BAL group was 61.4% in the diagnosis of lung cancer, which is much higher than with BAL alone. Rates of positive predictive values and negative predictive values in the combined group compared with the BALF alone group were 98.6% vs 100% and 47.6% vs 37.4%, respectively. Sensitivity in the BALF alone group was 12.5% in bronchoscopically invisible pulmonary lesions and as high as 52.1% in the combined group. CONCLUSION: The results from our study demonstrated that LBC of brushing samples could be used as an important complement of bronchoscopy and could have the potential to be widely applied.

Dissecting PCNA function with a systematically designed mutant library in yeast.

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sensitivity to DNA damaging reagents were identified using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.

Engineering the ribosomal DNA in a megabase synthetic chromosome.

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.