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Yakugaku Zasshi : Journal of the... 2024Quantitative NMR (qNMR), particularly H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for...
Quantitative NMR (qNMR), particularly H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the P nucleus, owing to the simplicity of its signals, and investigated the P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using P-qNMR agreed well with those for the established H-qNMR method in DO. In contrast, the SOF purity determined using P-qNMR was 1.6% higher than that for H-qNMR in the protic solvent CDOD. Therefore, using a protic solvent, such as CDOD, was not suitable for P-qNMR; the deuterium exchange with the RS for P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d was employed to determine the SOF purity. The data revealed that the SOF purities determined using P-qNMR agreed well with the established H-qNMR values, indicating that the absolute quantification of SOF using both P-qNMR and H-qNMR is possible in DMSO-d. Thus, we established an optimized and reproducible P-qNMR method in validation study across multiple laboratories.
Topics: Organophosphorus Compounds; Dimethyl Sulfoxide; Water; Solvents; Pharmaceutical Preparations
PubMed: 38556308
DOI: 10.1248/yakushi.23-00151-3 -
Journal of Infection in Developing... Jan 2024Human herpesvirus 6B (HHV-6B) encephalitis is common in immunosuppressed patients and presents a diagnostic challenge for physicians. Metagenomic next-generation...
Metagenomic Next-Generation Sequencing (mNGS) of cerebrospinal fluid for diagnosis of human herpesvirus 6B encephalitis following transplantation for severe aplastic anemia.
INTRODUCTION
Human herpesvirus 6B (HHV-6B) encephalitis is common in immunosuppressed patients and presents a diagnostic challenge for physicians. Metagenomic next-generation sequencing (mNGS) may facilitate early diagnosis of HHV-6B encephalitis. Herein, we described a case of HHV-6B encephalitis following transplantation for severe aplastic anemia (SAA) diagnosed by mNGS.
CASE SUMMARY
A 31-year-old male underwent myeloablative haploid hematopoietic stem cell transplantation for the treatment of SAA. On day + 21 after transplantation, the patient developed symptoms such as sudden epilepsy, drowsiness, memory dislocation, and memory loss. HHV-6B encephalitis was confirmed based on cranial MRI and mNGS of cerebrospinal fluid. Following antiviral therapy with sodium foscarnet, the symptoms improved and HHV-6B was negative by mNGS. There were no serious sequelae. Currently, the patient is in good health and is still under follow-up.
CONCLUSIONS
A case of HHV-6B encephalitis after SAA transplantation was diagnosed by mNGS of cerebrospinal fluid in time and was effectively treated with sodium foscarnet.
Topics: Male; Humans; Adult; Foscarnet; Herpesvirus 6, Human; Anemia, Aplastic; Encephalitis, Viral; Roseolovirus Infections; Hematopoietic Stem Cell Transplantation; Encephalitis; High-Throughput Nucleotide Sequencing; Sodium
PubMed: 38377081
DOI: 10.3855/jidc.18152 -
The Journal of International Medical... Jan 2024This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).
OBJECTIVE
This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).
METHODS
Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco's modified Eagle's medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined.
RESULTS
Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC.
CONCLUSIONS
Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.
Topics: Animals; Humans; Rats; Aorta; Foscarnet; Hyperphosphatemia; RNA, Small Interfering; Transcription Factors; Vascular Calcification; Sodium-Phosphate Cotransporter Proteins, Type III
PubMed: 38180904
DOI: 10.1177/03000605231222156 -
Chemical & Pharmaceutical Bulletin Jan 2024The spectrum of P-NMR is fundamentally simpler than that of H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative P-NMR...
The spectrum of P-NMR is fundamentally simpler than that of H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative P-NMR (P-qNMR) than using quantitative H-NMR (H-qNMR), which has been already established as an absolute determination method. We have previously reported a P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d were selected as the reference standards (RSs) for P-qNMR and H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CDOH was selected as the solvent for P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CDOD was the solvent of choice for the H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional H-qNMR for the determination of organophosphorus compounds.
Topics: Protons; Organophosphorus Compounds; Reference Standards; Water; Solvents
PubMed: 37899177
DOI: 10.1248/cpb.c23-00635 -
Journal of Clinical Virology : the... Oct 2023Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have...
BACKGROUND
Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have largely replaced viral culture for HSV diagnosis and molecular antiviral resistance testing is available for many viruses, HSV resistance testing continues to rely on phenotypic, viral culture-based methods, requiring weeks for results. Consequently, treatment of suspected HSV resistance remains largely empiric.
METHODS
We used HSV whole genome sequencing and a database of previously characterized HSV acyclovir and foscarnet resistance mutations to evaluate the performance of genotypic antiviral resistance testing among 19 control strains compared to in-house plaque reduction assay (PRA) and 25 clinical isolates sent for reference lab PRA antiviral resistance testing.
RESULTS
Among control strains, 23/29 (79.3%) results were concordant, 5 (17.2%) were indeterminate, and 1 (3.4%) was discordant. Indeterminate results were caused by variants of uncertain significance (VUS), including mutations without published phenotypes and mutations with contradictory results. Among clinical isolates, 14/40 (35%) results were concordant, 17 (42.5%) were indeterminate, and 9 (22.5%) were discordant. All discordant results were in reportedly phenotypically-susceptible HSV-1 strains yet possessed resistance mutations. Three contained resistant subpopulations. 6/8 (75%) discordant phenotypes were concordant with resistant genotypes upon repeat PRA.
CONCLUSIONS
These data support the combination of genotypic and phenotypic testing to diagnose HSV resistance more accurately and likely more rapidly than phenotypic testing alone. Genotypic context of resistance mutations and the ability of viral strains to form plaques in culture may affect phenotypic resistance results, highlighting the limitations of PRA alone as a gold standard method.
Topics: Humans; Antiviral Agents; Herpesvirus 2, Human; Acyclovir; Foscarnet; Herpesvirus 1, Human; Genotype; Drug Resistance, Viral; Herpes Simplex
PubMed: 37586184
DOI: 10.1016/j.jcv.2023.105554 -
Journal of Virology Aug 2023The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's...
The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.
Topics: Humans; Infant, Newborn; Cytidine Deaminase; Cytomegalovirus; DNA Replication; DNA, Viral; Epstein-Barr Virus Infections; Herpesviridae Infections; Herpesvirus 1, Human; Herpesvirus 4, Human; Immediate-Early Proteins; Minor Histocompatibility Antigens; Ribonucleotide Reductases; Viral Proteins; Virus Replication
PubMed: 37565748
DOI: 10.1128/jvi.00781-23 -
BMC Neurology May 2023Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human.
BACKGROUND
Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human.
CASE PRESENTATION
We report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss.
CONCLUSIONS
This case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.
Topics: Pseudorabies; Encephalitis, Viral; Endophthalmitis; Herpesvirus 1, Suid; Metagenomics; High-Throughput Nucleotide Sequencing; Delayed Diagnosis; Humans; Male; Middle Aged; Aqueous Humor; Acyclovir; Foscarnet; Methylprednisolone; Antiviral Agents; Blindness; DNA, Viral
PubMed: 37194001
DOI: 10.1186/s12883-023-03227-1 -
Mikrobiyoloji Bulteni Apr 2023Encephalitis is the inflammation of the brain parenchyma accompanied by mental or behavioral neurological dysfunction, sensory or motor deficits, speech or movement...
Encephalitis is the inflammation of the brain parenchyma accompanied by mental or behavioral neurological dysfunction, sensory or motor deficits, speech or movement disorders, and seizure. Encephalitis is an acute, life-threatening emergency that requires prompt recognition and a systematic approach for appropriate management. Human herpes virus (HHV-7) is one of the causative agents of encephalitis. In this report, a three years and six months old girl admitted to the hospital with the complaints of fever, cough, gushing vomiting, and altered consciousness, with fever, neck stiffness and blurred consciousness in her physical examination, and positive HHV-7 DNA polymerase chain reaction (PCR) in the cerebrospinal fluid (CSF) was presented. The CSF biochemistry of the patient was normal, and lymphocytic pleocytosis was detected in the CSF. Electroencephalography of the case revealed a cerebral dysfunction and hyperexcitability due to background activity abnormalities, and a cytotoxic transient lesion of the splenium in cranial magnetic resonance imaging. A 14-day foscarnet treatment was given to the patient after she progressed under empirical acyclovir treatment and HHV-7 was found to be the causative agent in the CSF. The patient was cured with the treatment and was followed up on an outpatient basis without any sequelae. In general, HHV-7 is estimated to be a common cause of pediatric acute encephalitis cases. It has been observed in the literature that almost all of the HHV-7-associated encephalitis cases occur after the age of six years, suggesting that HHV-7 causes neurological disease in children as a late infection. This case was three years and six months old and it was thought that she had encephalitis during primary infection. With this case report, we contributed to the literature by presenting a case of encephalitis in an immunocompetent pediatric patient with a transient splenial lesion associated with HHV-7, which progressed with empirical acyclovir treatment and responded to foscarnet treatment.
Topics: Humans; Child; Female; Infant; Antiviral Agents; Foscarnet; Herpesvirus 7, Human; Acyclovir; Encephalitis
PubMed: 37067214
DOI: 10.5578/mb.20239925 -
Transplantation and Cellular Therapy Jun 2023Human herpesvirus 6 (HHV-6) reactivation is common after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with higher mortality and... (Review)
Review
Human herpesvirus 6 (HHV-6) reactivation is common after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with higher mortality and increased transplantation-related complications. We hypothesized that preemptive treatment with a short course of foscarnet at a lower cutpoint of plasma HHV-6 viral load would be effective in treating early HHV-6 reactivation, preventing complications and precluding hospitalization of these patients. We reviewed outcomes of adult patients (age ≥18 years) who received preemptive treatment with once-daily foscarnet 60 to 90 mg/kg for 7 days for HHV-6 reactivation after allo-HSCT at our institution between May 2020 and November 2022. Plasma HHV-6 viral load was monitored by quantitative PCR twice monthly in the first 100 days post-transplantation and twice weekly after reactivation until resolution. Eleven patients with a median age of 46 years (range, 23 to 73 years) were included in the analysis. HSCT was performed with a haploidentical donor in 10 patients and with an HLA-matched related donor in 1 patient. The most common diagnosis was acute leukemia (9 patients). Myeloablative and reduced-intensity conditioning regimens were used in 4 and 7 patients, respectively. Ten of the 11 patients received post-transplantation cyclophosphamide-based graft-versus-host disease prophylaxis. The median follow-up was 440 days (range, 174 to 831 days), and the median time to HHV-6 reactivation was 22 days post-transplantation (range, 15 to 89 days). The median viral load at first reactivation was 3,100 copies/mL (range, 210 to 118,000 copies/mL), and the median peak viral load was 11,300 copies/mL (range, 600 to 983,000 copies/mL). All patients received a short course of foscarnet at either 90 mg/kg/day (n = 7) or 60 mg/kg/day (n = 4). In all patients, plasma HHV-6 DNA was undetectable at completion of 1 week of treatment. No HHV-6 encephalitis or pneumonitis occurred. All patients achieved neutrophil and platelet engraftment after a median of 16 days (range, 8 to 22 days) and 26 days (range, 14 to 168 days), respectively, with no secondary graft failure. No complications related to foscarnet administration were noted. One patient with very high HHV-6 viremia had recurrent reactivation and received a second course of foscarnet as an outpatient. A short course of once-daily foscarnet is effective in treating early HHV-6 reactivation post-transplantation and may reduce the incidence of HHV-6-related and treatment-related complications and preclude hospitalization in these patients.
Topics: Adult; Humans; Young Adult; Middle Aged; Aged; Adolescent; Foscarnet; Herpesvirus 6, Human; Hematopoietic Stem Cell Transplantation; Transplantation, Homologous; DNA, Viral
PubMed: 36878429
DOI: 10.1016/j.jtct.2023.02.022 -
BioRxiv : the Preprint Server For... Jan 2023The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses EBV and KSHV and the...
UNLABELLED
The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses EBV and KSHV and the alpha-herpesviruses HSV-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting involvement of an immediate early or early (IE-E) viral protein. In support of this mechanism, cycloheximide treatment of HCMV-infected cells prevents the expression of viral proteins and simultaneously blocks APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which is a viral DNA synthesis inhibitor affecting late protein expression, still permits A3B relocalization. These results combine to show that the beta-herpesvirus HCMV uses a fundamentally different, RNR-independent molecular mechanism to antagonize APOBEC3B.
IMPORTANCE
Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses in order to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.
PubMed: 36778493
DOI: 10.1101/2023.01.30.526383