Ensitrelvir in patients with SARS-CoV-2: A retrospective chart review.

Antivirals with proven effectiveness against the Omicron SARS-CoV-2 variant are required for COVID-19 treatment in hospitalized patients, particularly those with severe underlying conditions. Ensitrelvir, a 3C-like protease inhibitor, received emergency approval in Japan in November 2022, based on evidence of rapid symptom resolution in non-hospitalized patients, but confirmation of its effectiveness in hospitalized patients is lacking. This retrospective chart review reports outcomes for all patients who received ensitrelvir whilst hospitalized with SARS-CoV-2 infection at Rinku General Medical Center, Japan (November 2022-April 2023). Thirty-two hospitalized patients received 5 days of ensitrelvir treatment (375 mg loading dose, 125 mg as maintenance dose). Patients' mean age was 73.5 years and most had mild COVID-19. Patients exhibited various underlying diseases, most commonly hypertension (78.1%) and chronic kidney disease (25.0%). Seven (21.9%) patients were on hemodialysis. The most common concomitant medications were antihypertensives (59.4%) and corticosteroids (31.3%); 2 (6.3%) patients were being treated with rituximab; 28 (87.5%) patients had viral persistence following pre-treatment by remdesivir. Following ensitrelvir treatment, viral clearance was recorded in 18 (56.3%) patients by Day 6 and 25 (78.1%) patients at final measurement. All patients experienced clinical improvement as assessed by the investigator at Day 5. No intensive care unit admissions or deaths due to COVID-19 occurred. No new safety signals were observed. In conclusion, positive virological outcomes were observed following ensitrelvir treatment, in hospitalized patients with SARS-CoV-2 in a real-world setting, including high-risk patients, who failed previous antiviral therapy. These results require confirmation in more extensive studies. TRIAL REGISTRATION: UMIN000051300.

Functional Analyses of an Evolutionarily Conserved Acidic Patch on the Nucleosome.

The eukaryotic canonical nucleosome has an acidic patch on each H2A/H2B dimer. This acidic patch is also detected in histone variants, such as the H2A.Z (yeast Htz1)/H2B dimer. Here, we screened a comprehensive histone point mutant library and identified 11 histone residues located in four distinct nucleosome domains (Homologous Recombination (HR) Domain I-IV (HRD-I-IV)) with a potential role in HR. H2A-L66, -E93, and -L94 residues in HRD-I are located in the acidic patch region. Equivalent residues (H2A-L66 and Htz1-L73) partly compensate the function of each dimer. A common residue H2B-L109, which is located underneath of the acidic patch in both dimers, also partly compensates the function of each dimer. Upon exposure to DNA double-strand break (DSB)-inducing agents, the fragmented chromosomes of H2A-L66A mutant cells exhibited slow and limited recovery into intact chromosomes, suggesting that the H2A-L66A mutant is partly deficient in DSB repair. Furthermore, strand invasion, one of critical steps of HR, could be less efficient in H2A-L66A cells. All 11 HRD residues, including H2A-L66, are highly conserved in extant eukaryotic cells; therefore, our screening reported in this study will provide a foundation for future studies about the mechanisms underlying eukaryotic HR based on chromatin.

Transcription destabilizes centromere function.

Bi-oriented attachment of microtubules to the centromere is a pre-requisite for faithful chromosome segregation during mitosis. Budding yeast have point centromeres containing the cis-element proteins CDE-I, -II, and -III, which interact with trans-acting factors such as Cbf1, Cse4, and Ndc10. Our previous genetic screens, using a comprehensive library of histone point mutants, revealed that the TBS-I, -II, and -III regions of nucleosomes are required for faithful chromosome segregation. In TBS-III deficient cells, peri-centromeric nucleosomes containing the H2A.Z homolog Htz1 are lacking, however, it is unclear why chromosome segregation is defective in these cells. Here, we show that, in cells lacking TBS-III, both chromatin binding at the centromere and the total amount of some of the centromere proteins are reduced, and transcription through the centromere is up-regulated during M-phase. Moreover, the chromatin binding of Cse4, Mif2, Cbf1, Ndc10, and Scm3 was reduced upon ectopic transcription through the centromere in wild-type cells. These results suggest that transcription through the centromere displaces key centromere proteins and, consequently, destabilizes the interaction between centromeres and microtubules, leading to defective chromosome segregation. The identification of new roles for histone binding residues in TBS-III will shed new light on nucleosome function during chromosome segregation.

Functional Domain Mapping of Werner Interacting Protein 1 (WRNIP1).

Werner helicase-interacting protein 1 (WRNIP1) belongs to the AAA+ ATPase family and is conserved from Escherichia coli to human. In addition to an ATPase domain in the middle region of WRNIP1, WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain and two leucine zipper motifs in the N-terminal and C-terminal regions, respectively. Here, we report that the UBZ domain of WRNIP1 is responsible for the reduced levels of UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitylation in POLH-disrupted (polymerase η (Polη)-deficient) cells, and that the ATPase domain of WRNIP1 is involved in regulating the level of the PrimPol protein. The suppression of UV sensitivity of Polη-deficient cells by deletion of WRNIP1 was abolished by expression of the mutant WRNIP1 lacking the UBZ domain or ATPase domain, but not by the mutant lacking the leucine zipper domain in WRNIP1/POLH double-disrupted cells. The leucine zipper domain of WRNIP1 was required for its interaction with RAD18, a key factor in TLS (DNA translesion synthesis), and DNA polymerase δ catalytic subunit, POLD1. On the basis of these findings, we discuss the possible role of WRNIP1 in TLS.

Analysis of the molecular evolution of histone variant H2A.Z using a linker-mediated complex strategy and yeast genetic complementation.

The histone variant H2A.Z is deposited into chromatin by specific machinery and is required for genome functions. Using a linker-mediated complex strategy combined with yeast genetic complementation, we demonstrate evolutionary conservation of H2A.Z together with its chromatin incorporation and functions. This approach is applicable to the evolutionary analyses of proteins that form complexes with interactors.

An improved functional analysis of linker-mediated complex (iFALC) strategy.

The functional analysis of linker-mediated complex (FALC) strategy that facilitates functional analysis of a common subunit of multi-subunit protein complexes in cells constitutes three steps; (1) a common subunit is fused to a specific subunit via recombinant DNA, (2) mutation is introduced into a portion of the common subunit of the fused protein, and (3) the mutational effect on the fused protein is evaluated by transformation and analysis of multiple appropriate gene knockout yeast strains. Conceptually, the FALC strategy is applicable to any common subunit of multi-subunit protein complexes in any cell type. However, the proximity of two subunits to fuse, preparation of multiple gene knockout cells, and utilization of yeast cells can together prevent the practical and broad usage of the FALC strategy for analyzing all multi-subunit complexes in all cell types. In this study, we analyzed histone H2B as a common subunit of histone H2A/H2B and histone variant H2A.Z/H2B dimers. The FALC strategy was improved in three ways; (i) a long linker (up to 300 amino acids) was used to fuse H2B with H2A.Z in yeast cells, (ii) the effects of the fused H2B-H2A.Z harboring mutation in the H2B portion was evaluated in H2A.Z knockout yeast strains and it was not essential to knockout two copies of H2B genes, and (iii) this occurred even in vertebrate cells possessing a dozen H2B genes. This improved FALC (iFALC) strategy reveals that vertebrate H2B-D68, corresponding to yeast H2B-D71, is critical for chromatin binding of the H2A.Z/H2B dimer, and this is evolutionarily conserved.

WRNIP1 Controls the Amount of PrimPol.

Werner helicase-interacting protein 1 (WRNIP1) was originally identified as a protein that interacts with WRN, the product of the gene responsible for Werner syndrome. Our previous studies suggested that WRNIP1 is implicated in translesion synthesis (TLS), a process in which specialized TLS polymerases replace replicative DNA polymerase and take over DNA synthesis on damaged templates. We proposed that a novel error-free pathway involving DNA polymerase δ and primase-polymerase (PrimPol) functions to synthesize DNA on UV-damaged DNA templates in the absence of WRNIP1 and the TLS polymerase Polη. Hence, in the current study, we analyzed the relationship between WRNIP1 and PrimPol. We found that WRNIP1 and PrimPol form a complex in cells. PrimPol protein expression was reduced in cells overexpressing WRNIP1, but was increased in WRNIP1-depleted cells. The WRNIP1-mediated reduction in the amount of PrimPol was suppressed by treatment of the cells with proteasome inhibitors, suggesting that WRNIP1 is involved in the degradation of PrimPol via the proteasome.

Roles of common subunits within distinct multisubunit complexes.

Currently, there is no method to distinguish between the roles of a subunit in one multisubunit protein complex from its roles in other complexes in vivo. This is because a mutation in a common subunit will affect all complexes containing that subunit. Here, we describe a unique method to discriminate between the functions of a common subunit in different multisubunit protein complexes. In this method, a common subunit in a multisubunit protein complex is genetically fused to a subunit that is specific to that complex and point mutated. The resulting phenotype(s) identify the specific function(s) of the subunit in that complex only. Histone H2B is a common subunit in nucleosomes containing H2A/H2B or Htz1/H2B dimers. The H2B was fused to H2A or Htz1 and point mutated. This strategy revealed that H2B has common and distinct functions in different nucleosomes. This method could be used to study common subunits in other multisubunit protein complexes.

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na+-dependency, and kinetics of [3H]choline uptake were investigated. Effects of cationic drugs on the uptake of [3H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity. RESULTS: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [3H]Choline uptake occurred via a Na+-independent and pH-dependent transport system. The cells have two different [3H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [3H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [3H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression. CONCLUSIONS: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H+ antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.

Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts.

Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks.

Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

BACKGROUND: Interleukin (IL)-17A plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The expression of IL-17A in synovial mast cells (MCs) in RA and osteoarthritis (OA) has been reported, but the frequencies of IL-17A expression in synovial MCs have varied. The aim of this study was to investigate whether IL-17A expression is upregulated in human synovial MCs in RA and to elucidate the mechanism of IL-17A expression in synovial MCs. METHODS: Synovial tissues were obtained from patients with RA or OA undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Synovium-derived cultured MCs were generated by culturing synovial cells with stem cell factor. IL-17A expression was investigated using immunofluorescence in synovial tissues. IL-17A mRNA expression and its production from MCs were examined using RT-PCR and ELISA, respectively. RESULTS: The number of IL-17A-positive ((+)) synovial MCs and the percentage of IL-17A(+) MCs among all the IL-17A(+) cells from RA patients were not significantly increased compared with those from OA subjects. The synovium-derived cultured MCs spontaneously released small amounts of IL-17A. Neither IgE- nor IgG-dependent stimulation increased IL-17A production from the MCs. IL-33, tumor necrosis factor-α, C5a, lipopolysaccharide or IL-23 plus IL-1ß did not affect IL-17A production in MCs. CONCLUSIONS: The synovial MCs are not a main source of IL-17A in RA.

Tipin functions in the protection against topoisomerase I inhibitor.

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.

Tumor suppressor RecQL5 controls recombination induced by DNA crosslinking agents.

RecQ family DNA helicases function in the maintenance of genome stability. Mice deficient in RecQL5, one of five RecQ helicases, show a cancer predisposition phenotype, suggesting that RecQL5 plays a tumor suppressor role. RecQL5 interacts with Rad51, a key factor in homologous recombination (HR), and displaces Rad51 from Rad51-single stranded DNA (ssDNA) filaments in vitro. However, the precise roles of RecQL5 in the cell remain elusive. Here, we present evidence suggesting that RecQL5 is involved in DNA interstrand crosslink (ICL) repair. Chicken DT40 RECQL5 gene knockout (KO) cells showed sensitivity to ICL-inducing agents such as cisplatin (CDDP) and mitomycin C (MMC) and a higher number of chromosome aberrations in the presence of MMC than wild-type cells. The phenotypes of RECQL5 KO cells resembled those of Fanconi anemia gene KO cells. Genetic analysis using corresponding gene knockout cells showed that RecQL5 is involved in the FANCD1 (BRCA2)-dependent ICL repair pathway in which Rad51-ssDNA filament formation is promoted by BRCA2. The disappearance but not appearance of Rad51-foci was delayed in RECQL5 KO cells after MMC treatment. Deletion of Rad54, which processes the Rad51-ssDNA filament in HR, in RECQL5 KO cells increased sensitivity to CDDP and further delayed the disappearance of Rad51-foci, suggesting that RecQL5 and Rad54 have different effects on the Rad51-ssDNA filament. Furthermore, the frequency and variation of CDDP-induced gene conversion at the immunoglobulin locus were increased in RECQL5 KO cells. These results suggest that RecQL5 plays a role in regulating the incidence and quality of ICL-induced recombination.

Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation.

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere-specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule-depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty-three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS-I, -II, and -III (TBZ/benomyl-sensitive regions I-III). Elevation of mono-polar attachment induced by prior nocodazole treatment was observed in H2A-I112A (TBS-I), H2A-E57A (TBS-II), and H4-L97A (TBS-III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi-orientation, occurred in H2A-I112A and H2A-E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4-L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS-I and -II, are necessary for Sgo1-mediated chromosome bi-orientation and that TBS-III is required for Htz1 function.

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response.

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH(-/-)) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

Nucleosome surface containing nucleosomal DNA entry/exit site regulates H3-K36me3 via association with RNA polymerase II and Set2.

A nucleosome is composed of intrinsically disordered histone tails and a structured nucleosome core surrounded by DNA. A variety of modifiable residues on the intrinsically disordered histone tails have been identified in the last decade. Mapping of the functional residues on the structured nucleosome core surface was recently initiated by global analysis of a comprehensive histone point mutant library (histone-GLibrary). It stands to reason that a functional relationship exists between modifiable residues on the intrinsically disordered histone tails and functional residues on the structured nucleosome core; however, this matter has been poorly explored. During transcription elongation, trimethylation of histone H3 at lysine 36 (H3-K36me3) is mediated by histone methyltransferase Set2, which binds to RNA polymerase II. Here, we used a histone-GLibrary that encompasses the nucleosomal DNA entry/exit site to show that six residues (H2A-G107, H2A-I112, H2A-L117, H3-T45, H3-R49 and H3-R52) form a surface on the structured nucleosome core and regulate H3-K36me3. Trimethylation at H3-K4 introduced by histone methyltransferase Set1 was not affected by the mutation of any of the six residues. Chromatin immunoprecipitation analysis showed that most of these residues are critical for the chromatin association of RNA polymerase II and Set2, suggesting that these components regulate H3-K36me3 through functional interactions with the structured nucleosome core surface.

On the origin of the genetic code.

Mechanisms underlying how the genetic code was generated by Darwinian selection have remained elusive since the code was cracked in 1965. Here, I propose a hypothesis on the emergence of the genetic code and predict that its emergence was driven by sequential distinct selective pressures. According to the hypothesis, aminoacyl-RNAs for Glu, Asp, Lys, Tyr, His, Arg, Cys and Ser were first selected as cartridge-type subunits of three-subunit ribozymes. Aminoacyl-RNA subunits acting as cofactors were accommodated by the proto P-site of the large subunit of ribozymes. Importantly, I predict that there was no direct relationship between amino acids and codon and anticodon pairs. Duplication of the proto P-site could have created the proto A-site, enabling multi-subunit ribozymes to simultaneously interact with two-cartridge-type aminoacyl-RNA subunits. Random insertion of two cartridges would have instantly abolished enzymatic activity of multi-subunit ribozymes. On the other hand, if two tandemly aligned pairs of codons and anticodons specify two cartridges, dozens of different active pockets in multi-subunit ribozymes would have rapidly emerged, leading to the rise of extant organisms' metabolic pathways. The strong driving force of Darwinian selection described here could have created the primary genetic code for catalytic amino acids. Evolution of the protein translation system and events leading to the expansion of the genetic code until the time it was "frozen" are presented in detail.

The histone chaperone facilitates chromatin transcription (FACT) protein maintains normal replication fork rates.

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.

The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells.

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.

Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication.

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.