Background Metagenomic analysis targeting the 16S rRNA gene has made it possible to characterize the vast array of microorganisms contained in the gut. Aim The purpose of this study was to evaluate how gut microbiota change in intensive care unit (ICU) patients in the acute phase after admission. Methods This prospective observational cohort study investigated 12 patients admitted to a single ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated on admission. Fecal samples were collected from patients on days 1–2, 2–4, 5–8, and 7–10 after admission. DNA was extracted from fecal samples, and 16S rRNA deep sequencing was performed to monitor gut changes. Results Bacteria belonging to the phyla Firmicutes or Bacteroidetes were predominant in each sample. We observed serial dynamic changes in the percentages of Bacteroidetes and Firmicutes that were significantly altered during study period (p < 0.05). A ratio of Bacteroidetes to Firmicutes (B/F ratio) of >10 was seen in four of the six patients who died, whereas a B/F ratio of 10 or <0.10. There was a statistical difference in the B/F ratio between the dead patients and survivors (p = 0.022). Conclusions Dynamic changes in gut microbiota at the phylum level of ICU patients during the acute phase were identified by high-throughput DNA sequencing. An extreme imbalance in gut microbiota may be associated with prognosis in critically ill patients.

The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of “finished grade” because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

Combination immunotherapy with anti-programmed cell death1-ligand1 (PD-L1) and anti-vascular endothelial growth factor (VEGF) antibodies has become the standard treatment for patients with unresectable HCC (u-HCC). However, limited patients obtain clinical benefits. Cell-free DNA (cfDNA) in peripheral blood contains circulating tumor DNA (ctDNA) that reflects molecular abnormalities in tumor tissue. We investigated the potential of cfDNA/ctDNA as biomarkers for predicting the therapeutic outcome in u-HCC patients treated with anti-PD-L1/VEGF therapy. We enrolled a multicenter cohort of 85 HCC patients treated with atezolizumab and bevacizumab (Atezo/Bev) between 2020 and 2021. Pretreatment plasma was collected, and cfDNA levels were quantified. Ultradeep sequencing of cfDNA was performed with a custom-made panel for detecting mutations in 25 HCC-related cancer genes. We evaluated the association of cfDNA/ctDNA profiles and clinical outcomes. Patients with high plasma cfDNA levels showed a significantly lower response rate and shorter progression-free survival and overall survival (OS) than those with low cfDNA levels. ctDNA detected in 55% of HCC patients included the telomerase reverse transcriptase (TERT) promoter in 31% of these patients, tumor protein 53 (TP53) in 21%, catenin beta 1 (CTNNB1) in 13% and phosphatase and tensin homolog (PTEN) in 7%. The presence or absence of ctDNA did not predict the efficacy of Atezo/Bev therapy. Twenty-six patients with a TERT mutation had significantly shorter OS than those without. The presence of a TERT mutation and alpha-fetoprotein (AFP) ≥ 400 ng/mL were independent predictors of poor OS according to multivariate Cox proportional hazard analysis and could be used to stratify patients treated with Atezo/Bev therapy based on prognosis. In conclusion, pretreatment cfDNA/ctDNA profiling may be useful for predicting the therapeutic outcome in u-HCC patients treated with anti-PD-L1/VEGF therapy.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

Background The gut microbiota are reported to be altered in critical illness. The pattern and impact of dysbiosis on prognosis has not been thoroughly investigated in the ICU setting. Aims We aimed to evaluate changes in the gut microbiota of ICU patients via 16S rRNA gene deep sequencing, assess the association of the changes with antibiotics use or disease severity, and explore the association of gut microbiota changes with ICU patient prognosis. Methods Seventy-one mechanically ventilated patients were included. Fecal samples were collected serially on days 1–2, 3–4, 5–7, 8–14, and thereafter when suitable. Microorganisms of the fecal samples were profiled by 16S rRNA gene deep sequencing. Results Proportions of the five major phyla in the feces were diverse in each patient at admission. Those of Bacteroidetes and Firmicutes especially converged and stabilized within the first week from admission with a reduction in α-diversity (p < 0.001). Significant differences occurred in the proportional change of Actinobacteria between the carbapenem and non-carbapenem groups (p = 0.030) and that of Actinobacteria according to initial SOFA score and changes in the SOFA score (p < 0.001). An imbalance in the ratio of Bacteroidetes to Firmicutes within seven days from admission was associated with higher mortality when the ratio was > 8 or < 1/8 (odds ratio: 5.54, 95% CI: 1.39–22.18, p = 0.015). Conclusions Broad-spectrum antibiotics and disease severity may be associated with gut dysbiosis in the ICU. A progression of dysbiosis occurring in the gut of ICU patients might be associated with mortality.

The cell-cycle status of hematopoietic stem and progenitor cells (HSPCs) becomes activated following chemotherapy-induced stress, promoting bone marrow (BM) regeneration; however, the underlying molecular mechanism remains elusive. Here we show that BM-resident group 2 innate lymphoid cells (ILC2s) support the recovery of HSPCs from 5-fluorouracil (5-FU)–induced stress by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF). Mechanistically, IL-33 released from chemo-sensitive B cell progenitors activates MyD88-mediated secretion of GM-CSF in ILC2, suggesting the existence of a B cell–ILC2 axis for maintaining hematopoietic homeostasis. GM-CSF knockout mice treated with 5-FU showed severe loss of myeloid lineage cells, causing lethality, which was rescued by transferring BM ILC2s from wild-type mice. Further, the adoptive transfer of ILC2s to 5-FU–treated mice accelerates hematopoietic recovery, while the reduction of ILC2s results in the opposite effect. Thus, ILC2s may function by “sensing” the damaged BM spaces and subsequently support hematopoietic recovery under stress conditions.