Autologous bone marrow mononuclear cells to treat severe traumatic brain injury in children.

Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood-brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5-17 years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6 months and 12 months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1 year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of -20 600, 95% confidence interval (CI): -35 868 to -5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (-431 streamlines placebo versus -37 streamlines BMMNC; mean difference of -394, 95% CI: -803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics.

The Antibiotic Trimethoprim Displays Strong Mutagenic Synergy with 2-Aminopurine.

We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB-Rifr system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics.

When more is less: Emergent suppressive interactions in three-drug combinations.

BACKGROUND: In drug-drug interactions, there are surprising cases in which the growth inhibition of bacteria by a single antibiotic decreases when a second antibiotic is added. These interactions are termed suppressive and have been argued to have the potential to limit the evolution of resistance. Nevertheless, little attention has been given to suppressive interactions because clinical studies typically search for increases in killing efficiency and because suppressive interactions are believed to be rare based on pairwise studies. RESULTS: Here, we quantify the effects of single-, double-, and triple-drug combinations from a set of 14 antibiotics and 3 bacteria strains, totaling 364 unique three-drug combinations per bacteria strain. We find that increasing the number of drugs can increase the prevalence of suppressive interactions: 17% of three-drug combinations are suppressive compared to 5% of two-drug combinations in this study. Most cases of suppression we find (97%) are "hidden" cases for which the triple-drug bacterial growth is less than the single-drug treatments but exceeds that of a pairwise combination. CONCLUSIONS: We find a surprising number of suppressive interactions in higher-order drug combinations. Without examining lower-order (pairwise) bacterial growth, emergent suppressive effects would be missed, potentially affecting our understanding of evolution of resistance and treatment strategies for resistant pathogens. These findings suggest that careful examination of the full factorial of drug combinations is needed to uncover suppressive interactions in higher-order combinations.

Non-gadolinium dynamic angiography of the neurovasculature using arterial spin labeling MRI: preliminary experience in children.

OBJECTIVE: We demonstrate the potential clinical utility of a 4D non-gadolinium dynamic angiography technique based on arterial spin-labeling called contrast inherent inflow enhanced multi-phase angiography (CINEMA) in pediatric patients. MATERIALS AND METHODS: CINEMA was qualitatively compared to conventional time-of-flight (TOF) angiography in a cohort of 31 pediatric patients at 3 Tesla. RESULTS: CINEMA data were successfully acquired and reconstructed in all patients with no image artifacts. There were no cases where CINEMA was rated inferior to TOF in depicting intracranial vessel conspicuity. In 19 cases, CINEMA was rated equivalent to TOF and in the 12 remaining cases CINEMA was rated superior to TOF. CONCLUSION: There is a steadily rising concern in adults and children over the potential effects of intracranial deposition of gadolinium. CINEMA is therefore a viable alternative in dynamic neurovascular imaging.

Measurement of vertebral bone marrow proton density fat fraction in children using quantitative water-fat MRI.

OBJECTIVES: To investigate the feasibility of employing a 3D time-interleaved multi-echo gradient-echo (TIMGRE) sequence to measure the proton density fat fraction (PDFF) in the vertebral bone marrow (VBM) of children and to examine cross-sectional changes with age and intra-individual variations from the lumbar to the cervical region in the first two decades of life. MATERIALS AND METHODS: Quantitative water-fat imaging of the spine was performed in 93 patients (49 girls; 44 boys; age median 4.5 years; range 0.1-17.6 years). For data acquisition, a six-echo 3D TIMGRE sequence was used with phase correction and complex-based water-fat separation. Additionally, single-voxel MR spectroscopy (MRS) was performed in the L4 vertebrae of 37 patients. VBM was manually segmented in the midsagittal slice of each vertebra. Univariable and multivariable linear regression models were calculated between averaged lumbar, thoracic and cervical bone marrow PDFF and age with adjustments for sex, height, weight, and body mass index percentile. RESULTS: Measured VBM PDFF correlated strongly between imaging and MRS (R 2 = 0.92, slope = 0.94, intercept = -0.72%). Lumbar, thoracic and cervical VBM PDFF correlated significantly (all p < 0.001) with the natural logarithm of age. Differences between female and male patients were not significant (p > 0.05). CONCLUSION: VBM development in children showed a sex-independent cross-sectional increase of PDFF correlating with the natural logarithm of age and an intra-individual decrease of PDFF from the lumbar to the cervical region in all age groups. The present results demonstrate the feasibility of using a 3D TIMGRE sequence for PDFF assessment in VBM of children.

Improved fat-suppression homogeneity with mDIXON turbo spin echo (TSE) in pediatric spine imaging at 3.0 T.

Background Robust fat suppression remains essential in clinical MRI to improve tissue signal contrast, minimize fat-related artifacts, and enhance image quality. Purpose To compare fat suppression between mDIXON turbo spin echo (TSE) and conventional frequency-selective and inversion-recovery methods in pediatric spine MRI. Material and Methods Images from T1-weighted (T1W) and T2-weighted (T2W) TSE sequences coupled with conventional methods and the mDIXON technique were compared in 36 patients (5.8 ± 5.4 years) at 3.0 T. Images from 42 pairs of T1W (n = 16) and T2W (n = 26) scans were acquired. Two radiologists reviewed the data and rated images using a three-point scale in two categories, including the uniformity of fat suppression and overall diagnostic image quality. The Wilcoxon rank-sum test was used to compare the scores. Results The Cohen's kappa coefficient for inter-rater agreement was 0.69 (95% confidence interval [CI], 0.56-0.83). Images from mDIXON TSE were considered superior in fat suppression ( P < 0.01) in 22 (rater 1) and 25 (rater 2) cases, respectively. In 13 (rater 1) and 11 (rater 2) cases, mDIXON TSE demonstrated improved diagnostic image quality ( P < 0.01). In three cases, fat suppression was superior using inversion-recovery and likewise in one case mDIXON had poorer image diagnostic quality. Lastly, mDIXON and conventional fat-suppression methods performed similarly in 17 (rater 1) and 14 (rater 2) cases, and yielded equal diagnostic image quality in 28 (rater 1) and 30 (rater 2) cases. Conclusion Robust fat suppression can be achieved with mDixon TSE pediatric spine imaging at 3.0 T and should be considered as a permanent replacement of traditional methods, in particular frequency-selective techniques.

Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs.

UNLABELLED: We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10(-8)) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools. IMPORTANCE: Although mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.

Mutational Consequences of Ciprofloxacin in Escherichia coli.

We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif(r) system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics.

Increased signal intensities in the dentate nucleus and globus pallidus on unenhanced T1-weighted images: evidence in children undergoing multiple gadolinium MRI exams.

BACKGROUND: Recent reports have suggested residual gadolinium deposition in the brain in subjects undergoing multiple contrast-enhanced MRI exams. These findings have raised some concerns regarding gadolinium-based contrast agent (GBCA) usage and retention in brain tissues. OBJECTIVE: To summarize findings of hyperintense brain structures on precontrast T1-weighted images in 21 children undergoing multiple GBCA MRI exams. MATERIALS AND METHODS: This retrospective study involved 21 patients, each of whom received multiple MRI examinations (range: 5-37 exams) with GBCA over the course of their medical treatment (duration from first to most recent exam: 1.2-12.9 years). The patients were between 0.9 and 14.4 years of age at the time of their first GBCA exam. Regions of interest were drawn in the dentate nucleus and the globus pallidus on 2-D fast spin echo images acquired at 1.5 T. The signal intensities of these two structures were normalized by that of the corpus callosum genu. Signal intensity ratios from these patients were compared to control patients of similar ages who have never received GBCA. RESULTS: Signal intensity ratios increased between the first and the most recent MRI exam in all 21 patients receiving GBCA, with an increase of 18.6%±12.7% (range: 0.5% to 47.5%) for the dentate nucleus and 12.4%±7.4% (range: -1.2% to 33.7%) for the globus pallidus (P<0.0001). Signal intensity ratios were also higher in GBCA patients than in controls (P<0.01). The degree of signal intensity enhancement did not correlate with statistical significance to the cumulative number or volume of GBCA administrations each patient received, the patient's age or the elapsed time between the first and most recent GBCA MRI exams. CONCLUSION: These results in children are consistent with recent findings in adults, suggesting possible gadolinium deposition in the brain.

Exploring Synergy between Classic Mutagens and Antibiotics To Examine Mechanisms of Synergy and Antibiotic Action.

We used classical mutagens in Gram-negative Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. We used 4-nitroquinoline 1-oxide (4NQO), zebularine (ZEB), 5-azacytidine (5AZ), 2-aminopurine (2AP), and 5-bromodeoxyuridine (5BrdU) as mutagens (with bactericidal potency of 4NQO > ZEB > 5AZ > 2AP > 5BrdU) and vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL) as antibiotics. We detected the strongest synergies with 4NQO, an agent that oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics VAN, TMP, CPR, and GEN, but no synergies with the bacteriostatic antibiotics TET, ERY, and CHL. Each of the other mutagens displays synergies with the bactericidal antibiotics to various degrees that reflect their potencies, as well as with some of the other mutagens. The results support recent models showing that bactericidal antibiotics kill bacteria principally by ultimately generating more double-strand breaks than can be repaired. We discuss the synergies seen here and elsewhere as representing dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. We also used the results of pairwise tests to place the classic mutagens into functional antibacterial categories within a previously defined drug interaction network.

Synergistic interactions of vancomycin with different antibiotics against Escherichia coli: trimethoprim and nitrofurantoin display strong synergies with vancomycin against wild-type E. coli.

Gram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We used Escherichia coli mutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-type E. coli at concentrations of VAN as low as 6.25 µg/ml. This suggests that a very small percentage of exogenous VAN does enter E. coli but normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489-494, 2006, http://dx.doi.org/10.1038/ng1755).

Deoxycytidine deaminase-deficient Escherichia coli strains display acute sensitivity to cytidine, adenosine, and guanosine and increased sensitivity to a range of antibiotics, including vancomycin.

We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin.

Anatomy of a hotspot: Cisplatin hotspots in the tdk gene of Escherichia coli.

We previously reported that certain sub-regions of the thyA gene of Escherichia coli are more mutable than others when many different mutagens and mutators are analyzed (Mashiach et al., Mutation Research Fundamental Molecular Mechansims of Mutagenesis, 821: 111702, 2021). In this study, we focus on a single mutagen, cisplatin and verify that mutations occur preferentially at specific 3 bp sequences, but only when they appear in certain subregions of the gene. Moreover, we show that hotspots for some premutational lesions are camouflaged by the preferential repair effected by the uvrA,B,C-encoded excision repair system, even when they appear on the same strand. We do this by using a novel reporter gene in E. coli, the tdk gene that codes for thymidine deoxykinase, and we describe some of the advantages of utilizing this detection system.

An international study presenting a federated learning AI platform for pediatric brain tumors.

While multiple factors impact disease, artificial intelligence (AI) studies in medicine often use small, non-diverse patient cohorts due to data sharing and privacy issues. Federated learning (FL) has emerged as a solution, enabling training across hospitals without direct data sharing. Here, we present FL-PedBrain, an FL platform for pediatric posterior fossa brain tumors, and evaluate its performance on a diverse, realistic, multi-center cohort. Pediatric brain tumors were targeted due to the scarcity of such datasets, even in tertiary care hospitals. Our platform orchestrates federated training for joint tumor classification and segmentation across 19 international sites. FL-PedBrain exhibits less than a 1.5% decrease in classification and a 3% reduction in segmentation performance compared to centralized data training. FL boosts segmentation performance by 20 to 30% on three external, out-of-network sites. Finally, we explore the sources of data heterogeneity and examine FL robustness in real-world scenarios with data imbalances.

The Pediatric Neuroradiologist's Practical Guide to Capture and Evaluate Pre- and Postoperative Velopharyngeal Insufficiency.

Up to 30% of children with cleft palate will develop a severe speech disorder known as velopharyngeal insufficiency. Management of velopharyngeal insufficiency typically involves structural and functional assessment of the velum and pharynx by endoscopy and/or videofluoroscopy. These methods cannot provide direct evaluation of underlying velopharyngeal musculature. MR imaging offers an ideal imaging method, providing noninvasive, high-contrast, high-resolution imaging of soft-tissue anatomy. Furthermore, focused-speech MR imaging techniques can evaluate the function of the velum and pharynx during sustained speech production, providing critical physiologic information that supplements anatomic findings. The use of MR imaging for velopharyngeal evaluation is relatively novel, with limited literature describing its use in clinical radiology. Here we provide a practical approach to perform and interpret velopharyngeal MR imaging examinations. This article discusses the velopharyngeal MR imaging protocol, methods for interpreting velopharyngeal anatomy, and examples illustrating its clinical applications. This knowledge will provide radiologists with a new, noninvasive tool to offer to referring specialists.

Polynucleotide phosphorylase plays an important role in the generation of spontaneous mutations in Escherichia coli.

Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif(r) and the gyrB gene leading to Nal(r) and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.

Papillation in Bacillus anthracis colonies: a tool for finding new mutators.

Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria-Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair-deficient strains (MMR⁻) such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

The antiretroviral agents azidothymidine, stavudine, and didanosine have the identical mutational fingerprint in the rpoB region of Escherichia coli.

We looked at the mutational fingerprints of three antiretroviral (anti-HIV) agents, azidothymidine (AZT), stavudine (STAV), and didanosine (DIDA) in the rpoB system of Escherichia coli and compared them with each other and with the fingerprints of trimethoprim and of spontaneous mutations in a wild-type and a mutT background. All three agents gave virtually identical fingerprints in the wild-type background, causing only A:T→C:G changes at 3 of the 12 A:T→C:G possible sites among the total of 92 possible base substitution mutations, even though AZT and STAV are thymidine analogs but DIDA is an adenosine analog. As all three agents are reverse transcriptase inhibitors, and act as chain blockers, the common fingerprint may be a property of chain blocking agents.