Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA.

N6-methyladenosine (m6A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m6A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4+ T-cells or HIV-1 envelope protein treatment upregulates m6A levels of cellular RNA. Changes in the m6A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m6A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m6A-modified RNA from HIV-1-infected primary CD4+ T-cells based on immunoprecipitation. The enriched RNA with m6A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.

Antiretroviral Therapy Suppresses RNA N6-Methyladenosine Modification in Peripheral Blood Mononuclear Cells from HIV-1-Infected Individuals.

RNA N6-methyladenosine (m6A) modification is important for regulating gene expression and innate immune responses to viral infection. HIV-1 in vitro infection induces a significant increase in m6A modification of cellular RNA; however, whether m6A levels of cellular RNA are affected by HIV-1 replication or by antiretroviral therapy (ART) in infected individuals remains unknown. Using dot blot or enzyme-linked immunosorbent assay, we measured RNA m6A levels of peripheral blood mononuclear cells (PBMCs) from healthy donors or HIV-1-infected individuals with or without ART. Using a reverse transcription-quantitative polymerase chain reaction array, we quantified expression levels of 84 type-I interferon (IFN-I)-responsive genes in PBMCs from some individuals of these three groups. RNA m6A levels in PBMCs from HIV-1 viremic patients (n = 10) were significantly higher (p ≤ .0001) compared with ART-treated individuals (n = 22) or 1.5-fold higher compared with healthy donors (n = 14). However, the increase in RNA m6A levels did not correlate with changes in the expression of 10 m6A-regulatory genes. We found significant upregulation and downregulation in the expression of several IFN-I-responsive genes from HIV-1 viremic patients (n = 4) and ART-treated patients (n = 6) compared with healthy donors (n = 5), respectively. Our results suggest that post-transcriptional m6A modification may contribute to the regulation of IFN-I-responsive gene expression during HIV-1 infection and ART.

Functional Impacts of Epitranscriptomic m6A Modification on HIV-1 Infection.

Epitranscriptomic RNA modifications play a crucial role in the posttranscriptional regulation of gene expression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic RNA and plays a pivotal role in RNA fate. RNA m6A modification is regulated by a group of cellular proteins, methyltransferases (writers) and demethylases (erasers), which add and remove the methyl group from adenosine, respectively. m6A modification is recognized by a group of cellular RNA-binding proteins (readers) that specifically bind to m6A-modified RNA, mediating effects on RNA stability, splicing, transport, and translation. The functional significance of m6A modification of viral and cellular RNA is an active area of virology research. In this review, we summarize and analyze the current literature on m6A modification of HIV-1 RNA, the multifaceted functions of m6A in regulating HIV-1 replication, and the role of viral RNA m6A modification in evading innate immune responses to infection. Furthermore, we briefly discuss the future directions and therapeutic implications of mechanistic studies of HIV-1 epitranscriptomic modifications.

Epitranscriptomic N6-Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells.

N6-methyladenosine (m6A) is a dynamic posttranscriptional RNA modification that plays an important role in determining transcript fate. The functional consequence of m6A deposition is dictated by a group of host proteins that specifically recognize and bind the m6A modification, leading to changes in RNA stability, transport, splicing, or translation. The cellular m6A methylome undergoes changes during certain pathogenic conditions such as viral infections. However, how m6A modification of host cell transcripts and noncoding RNAs change during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection has not been reported. Here, we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. We identified mRNA and long and small noncoding RNA species that are differentially m6A modified in response to SARS-CoV-2 infection. The most significantly differentially methylated transcript was the precursor of microRNA-4486 (miRNA-4486), which showed significant increases in abundance and percentage of methylated transcripts in infected cells. Pathway analyses revealed that differentially methylated transcripts were significantly associated with several cancer-related pathways, protein processing in the endoplasmic reticulum, cell death, and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include several proteins involved in the type-I interferon response, inflammation, and cytokine signaling. IMPORTANCE Posttranscriptional modification of viral and cellular RNA by N6-methyladenosine (m6A) plays an important role in regulating the replication of many viruses and the cellular immune response to infection. We therefore sought to define the epitranscriptomic m6A profile of human lung epithelial cells infected with SARS-CoV-2. Our analyses demonstrate the differential methylation of both coding and noncoding cellular RNAs in SARS-CoV-2-infected cells compared to uninfected controls. Pathway analyses revealed that several of these RNAs may be involved in the cellular response to infection, such as type-I interferon. Our study implicates m6A modification of infected-cell RNA as a mechanism of posttranscriptional gene regulation during SARS-CoV-2 infection.

Translationally Controlled Tumor Protein-Mediated Stabilization of Host Antiapoptotic Protein MCL-1 Is Critical for Establishment of Infection by Intramacrophage Parasite Leishmania donovani.

In the early phase of infection, the intramacrophage pathogen Leishmania donovani protects its niche with the help of the antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Whether Leishmania could exploit MCL-1, an extremely labile protein, at the late phase is still unclear. A steady translational level of MCL-1 observed up to 48 h postinfection and increased caspase-3 activity in MCL-1-silenced infected macrophages documented its importance in the late hours of infection. The transcript level of MCL-1 showed a sharp decline at 6 h postinfection, and persistent MCL-1 expression in cyclohexamide-treated cells negates the possibility of de novo protein synthesis, thereby suggesting infection-induced stability. Increased ubiquitination, a prerequisite for proteasomal degradation of MCL-1, was also found to be absent in the late hours of infection. Lack of interaction with its specific E3 ubiquitin ligase MULE (MCL-1 ubiquitin ligase E3) and specific deubiquitinase USP9X prompted us to search for blockade of the ubiquitin-binding site in MCL-1. To this end, TCTP (translationally controlled tumor protein), a well-known binding partner of MCL-1 and antiapoptotic regulator, was found to be strongly associated with MCL-1 during infection. Phosphorylation of TCTP, a requirement for MCL-1 binding, was also increased in infected macrophages. Knockdown of TCTP decreased MCL-1 expression and short hairpin RNA-mediated silencing of TCTP in an infected mouse model of visceral leishmaniasis showed decreased parasite burden and induction of liver cell apoptosis. Collectively, our investigation revealed a key mechanism of how L. donovani exploits TCTP to establish infection within the host.

Improved loss-of-function CRISPR-Cas9 genome editing in human cells concomitant with inhibition of TGF-ß signaling.

Strategies to modulate cellular DNA repair pathways hold immense potential to enhance the efficiency of CRISPR-Cas9 genome editing platform. In the absence of a repair template, CRISPR-Cas9-induced DNA double-strand breaks are repaired by the endogenous cellular DNA repair pathways to generate loss-of-function edits. Here, we describe a reporter-based assay for expeditious measurement of loss-of-function editing by CRISPR-Cas9. An unbiased chemical screen performed using this assay enabled the identification of small molecules that promote loss-of-function editing. Iterative rounds of screens reveal Repsox, a TGF-ß signaling inhibitor, as a CRISPR-Cas9 editing efficiency enhancer. Repsox invariably increased CRISPR-Cas9 editing in a panel of commonly used cell lines in biomedical research and primary cells. Furthermore, Repsox-mediated editing enhancement in primary human CD4+ T cells enabled the generation of HIV-1-resistant cells with high efficiency. This study demonstrates the potential of transiently targeting cellular pathways by small molecules to improve genome editing for research applications and is expected to benefit gene therapy efforts.

SARS-CoV-2 spike E156G/Δ157-158 mutations contribute to increased infectivity and immune escape.

Breakthrough infections by emerging SARS-CoV-2 variants raise significant concerns. Here, we sequence-characterized the spike gene from breakthrough infections that corresponded to B.1.617 sublineage. Delineating the functional impact of spike mutations revealed that N-terminal domain (NTD)-specific E156G/Δ157-158 contributed to increased infectivity and reduced sensitivity to vaccine-induced antibodies. A six-nucleotide deletion (467-472) in the spike-coding region introduced this change in the NTD. We confirmed the presence of E156G/Δ157-158 from cases concurrently screened, in addition to other circulating spike (S1) mutations such as T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/Δ157-158 was present in more than 90% of the sequences reported from the USA and UK in October 2021. The spike-pseudotyped viruses bearing a combination of E156G/Δ157-158 and L452R exhibited higher infectivity and reduced sensitivity to neutralization. Notwithstanding, the post-recovery plasma robustly neutralized viral particles bearing the mutant spike. When the spike harbored E156G/Δ157-158 along with L452R and E484Q, increased cell-to-cell fusion was also observed, suggesting a combinatorial effect of these mutations. Our study underscores the importance of non-RBD changes in determining infectivity and immune escape.

SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles.

The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that the spike protein is better enriched in virions when the particles are produced in the presence of N protein. This enrichment of spike renders LV particles more infectious as well as less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Importantly, this improvement in infectivity is observed with both wild-type spike protein as well as the D614G mutant. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

From Entry to Egress: Strategic Exploitation of the Cellular Processes by HIV-1.

HIV-1 employs a rich arsenal of viral factors throughout its life cycle and co-opts intracellular trafficking pathways. This exquisitely coordinated process requires precise manipulation of the host microenvironment, most often within defined subcellular compartments. The virus capitalizes on the host by modulating cell-surface proteins and cleverly exploiting nuclear import pathways for post entry events, among other key processes. Successful virus-cell interactions are indeed crucial in determining the extent of infection. By evolving defenses against host restriction factors, while simultaneously exploiting host dependency factors, the life cycle of HIV-1 presents a fascinating montage of an ongoing host-virus arms race. Herein, we provide an overview of how HIV-1 exploits native functions of the host cell and discuss recent findings that fundamentally change our understanding of the post-entry replication events.

Adrenomedullin and Its Possible Role in Improved Survival in Female Patients with Sepsis: A Study in the South East Asian Region.

AIMS AND OBJECTIVES: Serum adrenomedullin (ADM) as a prognostic biomarker to study the gender-related differences in mortality pattern and its correlation with the sequential organ failure assessment (SOFA) and acute physiologic assessment and chronic health evaluation II (APACHE II) scores in patients of sepsis. MEASUREMENTS AND MAIN RESULTS: Eighty patients of sepsis of which 36 were males and 44 were females, were taken in the study as per sepsis III guidelines. They were followed up for a period of 28 days. Serum ADM was measured on day 1 and day 5. The endpoint was mortality or survival at day 28 after admission. The death rate among males was higher, with 23 of the total 36 (63.89%) patients having died when compared with females in which 25 patients out of 44 (56.82%) had died. The observed mortality rates correlated well with average APACHE II scores. The average APACHE II score was slightly higher in males (29 ± 8.97) when compared with females (27.02 ± 8.69). Similarly, day 1 SOFA and mean SOFA values were higher in males (10.22 ± 5.36) and (10.73 ± 6.01) when compared with females (8.27 ± 4.79) and (8.89 ± 5.6), respectively. Males despite having higher mortality rates, higher APACHE II, SOFA, and mean SOFA values were still having less mean levels of serum ADM (454.40 ± 81.13 pg/mL) when compared with females (479.62 ± 126.97 pg/mL). CONCLUSION: Adrenomedullin is a protective neurohormone with antibacterial and anti-inflammatory properties. It is elevated in all patients with sepsis but the rise is more so in the female when compared with males. Higher ADM levels in females may suggest the protective effect of ADM as a part of the general protective neurohormonal stress response, which may explain the low death rate in females in sepsis. HOW TO CITE THIS ARTICLE: Daga MK, Kumar L, Mawari G, Kumar N, Singh S, Mishra TK. Adrenomedullin and Its Possible Role in Improved Survival in Female Patients with Sepsis: A Study in the South East Asian Region. Indian J Crit Care Med 2020;24(12):1180-1184.

Polymorphism of 3' UTR of MAMLD1 gene is also associated with increased risk of isolated hypospadias in Indian children: a preliminary report.

OBJECTIVE: To study MAMLD1 gene polymorphisms, serum LH and testosterone levels amongst Indian children with isolated hypospadias (IH) and controls. MATERIALS AND METHODS: Screening of the MAMLD1 gene was performed by PCR sequencing method in 100 Indian children aged 0-12 years presenting with IH and 100 controls. LH and testosterone hormone levels were also assessed (categorized in four age-wise groups). RESULTS: IH subjects had significantly higher incidence of MAMLD1 polymorphism as compared to controls (33 vs 15 %, p = 0.01). Of various genomic variants identified in this study, the noteworthy novel ones were missense mutation P299A and single nucleotide polymorphism c.2960C>T in 3' UTR of Exon 7. While p 299A was found to cause protein structural instability consequent to amino acid change, eighty percent subjects with c.2960C>T in 3' UTR of Exon 7 (corresponding to newly discovered currently non-validated exon 11) were found to have lower testosterone levels when compared with their age group mean. IH showed statistically higher incidence of c.2960C>T in comparison to controls (22 vs 10 %, p value 0.046) and about 2.5-folds higher risk of this anomaly. CONCLUSION: Occurrence of MAMDL1 gene polymorphisms, specially of c.2960C>T in 3' UTR of its exon 7 is associated with a higher risk of IH in Indian children, probably by lowering androgenic levels.

The upside of failure: how regional student groups learn from their mistakes.

Success is the result of planning, hard work, determination, foresight, and a little bit of luck. Unfortunately, nobody has thought to pave the road to success. Although failure can be discouraging and time-consuming, it presents incredible learning opportunities-the biggest difference between those who succeed and those who abandon their projects lies in their response to adversity. This article reviews events undertaken by the Regional Student Groups (RSGs) in India and Argentina, the problems they encountered, and what can be learned from them. RSG-India attempted to organize an online scientific meeting (also known as a virtual conference) with geographically dispersed stakeholders, a totally new concept for them. RSG-Argentina tackled the challenge of organizing a two-day symposium, their first event ever. Some of the complications they faced were easy to fix, others led to the cancellation of activities, and all of them resulted in valuable lessons. The main goal of this article is to highlight, through their experiences, the universal importance of a healthy panel of contingency plans.

Hormonal profile in children with isolated hypospadias associates better with comprehensive score of local anatomical factors as compared to meatal location or degree of chordee.

BACKGROUND: To evaluate if hormonal profile of children with isolated hypospadias (IH) associates better with comprehensive local anatomical factor score (LAFS) than with clinically adjudged urethral meatus location or severity of chordee/k.j. MATERIAL AND METHODS: Ninety-nine children with IH were enrolled, as per inclusion criteria. Meatal location was recorded at first clinical examination in OPD; while LAFS was computed per-operatively using indigenously devised scale, except for neonates. Hypospadiacs were first classified into three standard meatal based groups and subsequently into LAFS based two groups (≤19, >19). For all participants, pre HCG and post HCG (96 hour post- injection) estimation of serum gonadotropins, DHEA-S, estrogen (E), progesterone (P), testosterone (T) and Dihydrotestosterone (DHT) was done. Statistical tests were applied to assess significance of hormonal levels with respect to meatal location, chordee and LAFS. RESULTS: Only FSH levels differed significantly among meatal based groups; while among LAFS groups, multiple hormonal differences were noted; with poor LAFS associated significantly with higher FSH, LH and lower E, T/DHT. Children with severe degree of chordee had poorer T output and a significantly lower LAFS as compared to those with moderate/mild chordee. CONCLUSION: Serotoli cell dysfunction, indirectly indicated by high FSH was found among midpenile hypospadiacs and those with poorer LAFS. Since groups based on LAFS revealed multiple intergroup hormonal differences than what was seen for meatal/chordee based groups; LAFS should be considered a better guide for prognostication and for deciding about hormonal supplementation. Lower androgenic output was particularly noted in children with severe chordee.

Building the future of bioinformatics through student-facilitated conferencing.

Sharing results, techniques, and challenges is paramount to advance our understanding of any field of science. In the scientific community this exchange of ideas is mainly made possible through national and international conferences. Scientists have the opportunity to showcase their work, receive feedback, and improve their presentation skills. However, conferences can be large and intimidating for young researchers. In addition, for many of the more prestigious conferences, the very high number of submissions and low selection rate are major limitations to aspiring young researchers aiming to present their work to the scientific community. To improve student participation and proliferation of information, regional student groups have successfully organized conferences and symposia specifically aimed at students. This gives more students the opportunity to present their work and receive valuable experience and insight from peers and leaders in the field. At the same time, it is an ideal way for students to gain familiarity with the conference experience. In this paper, we highlight some of the benefits of participating in such student conferences, and we review the challenges we have encountered when organizing them. Both topics are illustrated in detail with examples from different ISCB Student Council Regional Student Groups.

Interaction of low-frequency axisymmetric ultrasonic guided waves with bends in pipes of arbitrary bend angle and general bend radius.

The use of ultrasonic guided waves for the inspection of pipes with elbow and U-type bends has received much attention in recent years, but studies for more general bend angles which may also occur commonly, for example in cross-country pipes, are limited. Here, we address this topic considering a general bend angle φ, a more general mean bend radius R in terms of the wavelength of the mode studied and pipe thickness b. We use 3D Finite Element (FE) simulation to understand the propagation of fundamental axisymmetric L(0,2) mode across bends of different angles φ. The effect of the ratio of the mean bend radius to the wavelength of the mode studied, on the transmission and reflection of incident wave is also considered. The studies show that as the bend angle is reduced, a progressively larger extent of mode-conversion affects the transmission and velocity characteristics of the L(0,2) mode. However the overall message on the potential of guided waves for inspection and monitoring of bent pipes remains positive, as bends seem to impact mode transmission only to the extent of 20% even at low bend angles. The conclusions seem to be valid for different typical pipe thicknesses b and bend radii. The modeling approach is validated by experiments and discussed in light of physics of guided waves.

Breaking the ice and forging links: the importance of socializing in research.

When meeting someone for the first time-whether another PhD student, or the Founding Editor-in-chief of PLOS Computational Biology-nothing breaks the ice like eating pancakes or having drinks together. A social atmosphere provides a relaxed, informal environment where people can connect, share ideas, and form collaborations. Being able to build a network and thrive in a social environment is crucial to a successful scientific career. This article highlights the importance of bringing people together who speak the same scientific language in an informal setting. Using examples of events held by Regional Student Groups of the ISCB's Student Council, this article shows that socializing is much more than simply sharing a drink.

Children with isolated hypospadias have different hormonal profile compared to those with associated anomalies.

AIM: The objective of this study is to compare the hormonal profile of children with isolated hypospadias to controls and hypospadiacs with associated anomalies. STUDY DESIGN: Prospective observation at a tertiary referral hospital. STUDY SUBJECTS: One hundred consecutive children (0-12 years) with isolated hypospadias (H), 23 with hypospadias and associated anomalies (HO). CONTROLS: One hundred children (0-12 years) without any genitourinary/endocrine abnormalities (C). PROCEDURE: Prehuman chorionic gonadotropin (HCG) and post-HCG fasting blood samples were drawn for estimation of serum gonadotropins, dehydroepiandrosterone sulfate (DHEA-S), estrogen (E), progesterone (P), and testosterone (T) and dihydrotestosterone (DHT). STATISTICAL ANALYSIS: Differences in hormonal levels between controls and subjects were computed with p < or = 0.05 as significant. RESULTS: Compared with controls, "H" had significantly higher follicular stimulating hormone (FSH) (1.37 vs. 1.29 mIU/mL p=0.01), lower estrogen (8.08 vs. 13.78 pg/mL, p=0.00), and lower DHEA-S (27.34 vs. 40.24 microg/dL, p=0.03) levels; HO had higher FSH, lower basal T (0.13 vs. 0.46 ng/mL, p=0.01), and lower peak testosterone (1.53 vs. 2.32 ng/mL, p=0.01). "HO" had lower androgens (basal T, 0.13 vs. 0.29 ng/mL, p=0.03; peak T, 1.53 vs. 2.36 ng/mL, p=0.01), and higher estrogen (12.56 vs. 8.08 pg/mL, p=0.001) and progesterone (0.46 vs. 0.31 ng/mL, p=0.04) levels in comparison with H. CONCLUSION: Consistently lower output of androgens among HO explains the association of other anomalies (generally undescended testes) in them. High FSH among hypospadiacs hints at the possibility of Sertoli cell dysfunction and may have long-lasting sequelae for reproductive functions during adulthood. However, Leydig cell functions are affected more among HO.

Breast cancer: interaction between oxidant-antioxidant dynamics and inflammation in Indian females.

OBJECTIVES: Oxidative stress and pro-inflammatory mediators have been implicated in breast carcinogenesis. We attempted to evaluate the markers of oxidative stress, antioxidant mechanism and the inflammatory pathway in patients with breast cancer. METHODS: This study was carried out in departments of Biochemistry and Surgery, Maulana Azad Medical College and associated Hospitals, New Delhi, India. A total of 60 cases of carcinoma of the breast and 60 healthy controls were included in the study. The parameters that were assayed include markers of oxidative stress-conjugated dienes, thiobarbitone reactive substances (TBARS), antioxidants-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH) and markers of inflammation-interleukin-6(IL-6) and ferritin. RESULTS: There was a significant decline in the antioxidant levels and a significant rise in oxidant levels in patients with carcinoma of the breast, compared to controls. The inflammatory markers-IL-6 and ferritin-were also significantly higher in patients with breast cancer. A significantly positive correlation was observed between the IL-6 levels and conjugated dienes with the stage of breast carcinoma; whilst a significantly negative correlation was observed between the levels of conjugated dienes and superoxide dismutase and superoxide dismutase levels with the disease staging. CONCLUSIONS: This study underlines the interplay between inflammatory pathways and oxidative stress in the pathogenesis of breast cancer. MINI ABSTRACT: An intense research is underway to identify the possible risk factors and the molecular mechanisms involved in pathogenesis of breast cancer. Inflammation and oxidative stress are two such etiologies investigated in our study.

Pyrazinamide blood concentrations in children suffering from tuberculosis: a comparative study at two doses.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Pyrazinamide is recommended in doses varying from 15 to 40 mg kg(-1). The most commonly used average daily dose is 25 mg kg(-1). Its use is associated with dose dependent hepatotoxicity. Lower doses are not used because of lack of pharmacokinetic data especially in children. There is only one detailed study of pyrazinamide in children at a dose of 35 mg kg(-1). WHAT THIS STUDY ADDS: This is the first study evaluating serum concentrations of pyrazinamide in children at a dose of 15 mg kg(-1) which is on the lower side of the recommended dose. The study also compared the serum concentrations and pharmacokinetics achieved with this dose with the widely used dose of 25 mg kg(-1) in children suffering from tuberculosis. The pharmacokinetics and pharmacodynamic indices of pyrazinamide were comparable with the 25 and 15 mg kg(-1) doses. AIMS: To evaluate the pharmacokinetics and pharmacodynamic indices of pyrazinamide at doses of 15 and 25 mg kg(-1) in children suffering from tuberculosis. METHODS: Twenty children with tuberculosis received pyrazinamide at a single dose of 25 mg kg(-1) (group I) and 15 mg kg(-1) (group II). Serial blood samples were collected and the drug concentrations were analyzed spectrophotometrically. The pharmacokinetic parameters were calculated and the duration of time for which pyrazinamide concentrations in serum remained above the pyrazinamide inhibitory concentrations of 20 microg ml(-1) and 25 microg ml(-1) was studied. RESULTS: The mean peak serum concentration was 42.4 +/- 3.3 microg ml(-1) (95% CI +/- 6.5) and 38.6 +/- 3.9 microg ml(-1) (95% CI +/- 7.7) in groups I and II, respectively. The elimination half-life was 9.3 +/- 1.3 h and 10.5 +/- 2.3 h (P = 0.6) and clearance was 0.06 +/- 0.01 l h(-1) kg(-1) and 0.04 +/- 0.01 l h(-1) kg(-1) (P = 0.08) in groups I and II, respectively. Pharmacokinetic parameters and PKPD indices were comparable with both the doses. CONCLUSIONS: The study indicates that comparable serum concentrations of pyrazinamide are attained with 25 mg kg(-1) and 15 mg kg(-1) doses in children. The elimination half-life was longer and volume of distribution greater in children than in the adult population.