Weiyi Li

High altitudes pose extreme survival challenges for organisms, yet the origins and molecular strategies underlying their resilience remain poorly understood. Here, we report the molecular and evolutionary mechanisms underlying stress resilience in Apourosomoida sp. LHA081A01, a ciliate isolated from a high-altitude Tibetan salt lake that endures high salinity, low temperature, and hypoxia. We identified TreT glycosyltransferases, acquired through horizontal gene transfer from an anaerobic and halophilic Desulfobacteraceae bacterium, to be involved in the synthesis of α,α-trehalose—a universal protein stabilizer absent in most other ciliates but essential for counteracting multiple environmental stressors. Additional strategies include β-carotene accumulation to mitigate oxidative stress from hypoxia, along with numerous others common to many eukaryotes. Extensive gene family expansions and rapid divergence of stress‑responsive genes underscore their evolutionary significance and critical role in surviving harsh habitats. Intolerance to low salinity may render this ciliate, and other protists, vulnerable to climate‑driven salinity declines in Tibetan salt lakes. Together, these extraordinary features—shaped by horizontal gene transfer, natural selection, and regulatory plasticity—position high-altitude microbial eukaryotes as powerful extremophile models for uncovering the molecular mechanisms of stress resilience and adaptive evolution across life.

Although transcript-error rates are markedly higher than DNA-level mutation rates, a broad perspective on the degree to which they diverge across lineages remains to be developed. Using modified rolling-circle sequencing, we found a narrow range of transcript-error rates across the Tree of Life, with little evidence supporting local control of error rates associated with gene expression levels. Most errors result in missense changes if translated, and, as with a fraction of nonsense errors, these are underrepresented relative to random expectations, suggesting the existence of mechanisms for purging some such errors. To understand how natural selection and random genetic drift might shape transcript-error rates, we present a model based on cell biology and population genetics. However, while this framework helps understand the evolution of this highly conserved trait, as currently structured, it explains only 20% of the variation in the data, suggesting a need for further theoretical work in this area.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45 000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

The expression of genomically-encoded information is not error-free. Transcript-error rates are dramatically higher than DNA-level mutation rates, and despite their transient nature, the steady-state load of such errors must impose some burden on cellular performance. However, a broad perspective on the degree to which transcript-error rates are constrained by natural selection and diverge among lineages remains to be developed. Here, we present a genome-wide analysis of transcript-error rates across the Tree of Life using a modified rolling-circle sequencing method, revealing that the range in error rates is remarkably narrow across diverse species. Transcript errors tend to be randomly distributed, with little evidence supporting local control of error rates associated with gene-expression levels. A majority of transcript errors result in missense errors if translated, and as with a fraction of nonsense transcript errors, these are underrepresented relative to random expectations, suggesting the existence of mechanisms for purging some such errors. To quantitatively understand how natural selection and random genetic drift might shape transcript-error rates across species, we present a model based on cell biology and population genetics, incorporating information on cell volume, proteome size, average degree of exposure of individual errors, and effective population size. However, while this model provides a framework for understanding the evolution of this highly conserved trait, as currently structured it explains only 20% of the variation in the data, suggesting a need for further theoretical work in this area.

Errors can occur at any level during the replication and transcription of genetic information. Genetic mutations derived mainly from replication errors have been extensively studied. However, fundamental details of transcript errors, such as their rate, molecular spectrum, and functional effects, remain largely unknown. To globally identify transcript errors, we applied an adapted rolling-circle sequencing approach to Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, and Mesoplasma florum, revealing transcript-error rates 3 to 4 orders of magnitude higher than the corresponding genetic mutation rates. The majority of detected errors would result in amino-acid changes, if translated. With errors identified from 9929 loci, the molecular spectrum and distribution of errors were uncovered in great detail. A G→A substitution bias was observed in M. florum, which apparently has an error-prone RNA polymerase. Surprisingly, an increased frequency of nonsense errors towards the 3′ end of mRNAs was observed, suggesting a Nonsense-Mediated Decay-like quality-control mechanism in prokaryotes.

The number of lymph node dissections (LNDs) is an independent factor influencing the survival time of patients with bladder cancer (BCa) after radical resection (RC). The present study aimed to investigate the association between the number of LNDs and the survival of patients with BCa at different stages and who underwent RC in the United States of America and China. Records from 17,730 American patients with BCa and 158 Chinese patients with BCa were collected from the Surveillance, Epidemiology and End Results (SEER) and the Shanghai Tenth People's Hospital (China) databases, respectively. Kaplan‑Meier curve and χ2 test were used to determine the overall survival time (OS) of patients with BCa. Cox regression analysis was used to analyze the effects of LND number on OS. Overall, 13,421 (75.7%) patients were negative for lymph node metastasis (N0) and 4,309 (24.3%) were positive for lymph node metastasis (N+) among the 17,730 American patients with BCa. In the group of 158 Chinese patients, 125 (79.1%) were N0 and 33 (20.9%) were N+. In the American patients, the median number of dissected nodes was 11.0 [interquartile range (IQR)=3.0‑21.0] for N0 patients and 14.0 (IQR=8.0‑23.0) for N+ patients. The median number of LNDs was 5.0 (IQR=2.0‑7.0) for Chinese N0 patients and 5.0 (IQR=1.5‑10.5) for Chinese N+ patients. The number of LNDs may therefore be an independent factor associated with survival in patients who underwent RC. Furthermore, a higher number of LNDs was associated with longer OS in patients with BCa [American patients, ≥13 nodes vs. ≤5 nodes, hazard ratio (HR)=0.62, 95% confidence interval (CI)=0.59‑0.65, P<0.001; Chinese patients, ≥5 nodes vs. ≤1 node, HR=0.27, 95% CI=0.12‑0.62, P=0.002]. The number of LNDs in patients with BCa and N+ was higher compared with number of LNDs in patients with BCa and N0 who underwent RC. More extensive LND improved the OS in both the patients from USA and China. Increasing the number of LNDs may therefore be crucial when treating patients with BCa.

Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes meningitis in humans and pigs. However, the mechanism whereby SS2 crosses the microvasculature endothelium of the brain is not understood. In this study, transposon (TnYLB‐1) mutagenesis was used to identify virulence factors potentially associated with invasive ability in pathogenic SS2. A poorly invasive mutant was identified, and was found to contain a TnYLB‐1 insertion in the serine/threonine kinase (stk) gene. Transwell chambers containing hBMECs were used to model the blood‐brain barrier (BBB). We observed that the SS2 wild‐type ZY05719 strain crossed the BBB model more readily than the mutant strain. Hence, we speculated that STK is associated with the ability of crossing blood‐brain barrier in SS2. In vitro, compared with ZY05719, the ability of the stk‐deficient strain (Δstk) to adhere to and invade both hBMECs and bEnd.3 cells, as well as to cross the BBB, was significantly attenuated. Immunocytochemistry using antibodies against claudin‐5 in bEnd.3 cells showed that infection by ZY05719 disrupted BBB tight junction proteins to a greater extent than in infection by Δstk. The studies revealed that SS2 initially binds at or near intercellular junctions and crosses the BBB via paracellular traversal. Claudin‐5 mRNA levels were indistinguishable in ZY05719‐ and Δstk‐infected cells. This result indicated that the decrease of claudin‐5 was maybe induced by protein degradation. Cells infected by ZY05719 exhibited higher ubiquitination levels than cells infected by Δstk. This result indicated that ubiquitination was involved in the degradation of claudin‐5. Differential proteomic analysis showed that E3 ubiquitin protein ligase HECTD1 decreased by 1.5‐fold in Δstk‐infected bEnd.3 cells relative to ZY05719‐infected cells. Together, the results suggested that STK may affect the expression of E3 ubiquitin ligase HECTD1 and subsequently increase the degradation of claudin‐5, thus enabling SS2 to traverse the BBB.

This paper provides the first comprehensive analysis of the fidelity of transcription in eukaryotic cells.