Computational Methods Reveal a Series of Cyclic and Linear Lichenysins and Surfactins from the Vietnamese Marine Sediment-Derived Streptomyces Strain G222.

The Streptomyces strain G222, isolated from a Vietnamese marine sediment, was confidently identified by 16S rRNA gene sequencing. Its AcOEt crude extract was successfully analyzed using non-targeted LC-MS/MS analysis, and molecular networking, leading to a putative annotation of its chemical diversity thanks to spectral libraries from GNPS and in silico metabolite structure prediction obtained from SIRIUS combined with the bioinformatics tool conCISE (Consensus Annotation Propagation of in silico Elucidations). This dereplication strategy allowed the identification of an interesting cluster of a series of putative cyclic and linear lipopeptides of the lichenysin and surfactin families. Lichenysins (3-7) were isolated from the sub-fraction, which showed significant anti-biofilm activity against Pseudomonas aeruginosa MUC-N1. Their structures were confirmed by detailed 1D and 2D NMR spectroscopy (COSY, HSQC, HMBC, TOCSY, ROESY) recorded in CD3OH, and their absolute configurations were determined using the modified Marfey's method. The isolated lichenysins showed anti-biofilm activity at a minimum concentration of 100 µM. When evaluated for antibacterial activity against a panel of Gram-positive and Gram-negative strains, two isolated lichenysins exhibited selective activity against the MRSA strain without affecting its growth curve and without membranotropic activity. This study highlights the power of the MS/MS spectral similarity strategy using computational methods to obtain a cross-validation of the annotated molecules from the complex metabolic profile of a marine sediment-derived Streptomyces extract. This work provides the first report from a Streptomyces strain of combined cyclic and linear lichenysins and surfactins, known to be characteristic compounds of the genus Bacillus.

Environmental contamination with Clostridioides (Clostridium) difficile in Vietnam.

AIMS: To investigate the prevalence, molecular type, and antimicrobial susceptibility of Clostridioides difficile in the environment in Vietnam, where little is known about C. difficile. METHODS AND RESULTS: Samples of pig faeces, soils from pig farms, potatoes, and the hospital environment were cultured for C. difficile. Isolates were identified and typed by polymerase chain reaction (PCR) ribotyping. The overall prevalence of C. difficile contamination was 24.5% (68/278). Clostridioides difficile was detected mainly in soils from pig farms and hospital soils, with 70%-100% prevalence. Clostridioides difficile was isolated from 3.4% of pig faecal samples and 5% of potato surfaces. The four most prevalent ribotypes (RTs) were RTs 001, 009, 038, and QX574. All isolates were susceptible to metronidazole, fidaxomicin, vancomycin, and amoxicillin/clavulanate, while resistance to erythromycin, tetracycline, and moxifloxacin was common in toxigenic strains. Clostridioides difficile RTs 001A+B+CDT- and 038A-B-CDT- were predominantly multidrug resistant. CONCLUSIONS: Environmental sources of C. difficile are important to consider in the epidemiology of C. difficile infection in Vietnam, however, contaminated soils are likely to be the most important source of C. difficile. This poses additional challenges to controlling infections in healthcare settings.

Effect of ischemic time on pediatric heart transplantation outcomes: is it the same for all allografts?

BACKGROUND: Studies have shown that the optimal ischemia time (IT) threshold in pediatric heart transplantation (PHT) is up to 4 h, independent of other donor organ factors. The purpose of this study was to examine the relationship between IT and donor left ventricular ejection fraction (LVEF) and study their impact on PHT outcomes. METHODS: This is a retrospective cohort study of PHT (<18 years) identified in UNOS between January 2000 and March 2020. Post-transplantation survival analysis of patients receiving donor hearts with IT<4, 4-6, and >6 h was performed using Kaplan-Meier curves. Cohort was divided according to donor LVEF median value, and survival was analyzed. Cox regression was performed. RESULTS: Median LVEF was 65% in the study cohort (6669 PHT). Overall, IT>6 h was associated with worse survival compared to <4 h regardless of donor LVEF. For allografts with LVEF < 65%, IT = 4-6 h was associated with worse survival compared with IT < 4 h (p = .006) but had similar survival compared with IT > 6 h (p = .315). For allografts with LVEF ≥ 65%, IT = 4-6 h had similar survival compared with <4 h (p = .175) but improved survival compared with >6 h (p = .003). After adjusting for donor and recipient variables, Cox regression showed that IT = 4-6 h was not associated with increased mortality for LVEF ≥ 65%. CONCLUSIONS: The IT threshold of 4 h does not apply to all allografts. Recipients of hearts with LVEF≥65% can tolerate an IT up to 6 h without any detriment to survival. Routine acceptance of these donor hearts could mitigate longer waiting times and poor donor availability for many candidates.

Clostridioides (Clostridium) difficile in children with diarrhoea in Vietnam.

BACKGROUND: Clostridioides (Clostridium) difficile commonly causes hospital-acquired infection which can range from mild diarrhoea to life-threatening toxic megacolon and even death. Reports on C. difficile infection (CDI) in Vietnam are limited, so this study was designed to evaluate the prevalence, molecular epidemiology and antimicrobial susceptibility of C. difficile isolated from children with diarrhoea in Vietnam. Infants are often colonised with C. difficile and it was hypothesised that those colonising strains would represent strains of C. difficile circulating in the hospital/region at the time, however, this was not an attempt to determine if C. difficile was the cause of the diarrhoea. METHODS: Diarrhoeal stool samples collected at two children's hospitals in northern Vietnam from October 1, 2020 to February 28, 2021 were transported to Perth, Western Australia, for culture of C. difficile and further investigations on isolates; PCR ribotyping, toxin gene profiling and antimicrobial susceptibility testing. RESULTS: From these hospitals, 370 diarrhoeal stool samples were collected, most from children aged 1-15 months (71.9%; 266/370). The overall prevalence of C. difficile in stool samples from children aged ≤16 years was 37.8% (140/370) and the highest prevalence was in the 2-12 months age group (52.9%; 74/140). In total, 151 isolates of C. difficile were recovered; the proportion of toxigenic isolates was 16.6% (25/151). Of the 25 toxigenic C. difficile isolates, the toxin gene profiles A+B+CDT- and A-B+CDT- comprised 72% and 28%, respectively. The four most prevalent C. difficile ribotypes (RTs) were QX 011 (25/151), RT 010 (25/151), QX 107 (12/151) and RT 012 (11/151). All isolates were susceptible to vancomycin, metronidazole and fidaxomicin, while there was significant resistance to clindamycin (90.1%), and some to moxifloxacin (6.6%) and rifaximin (3.3%). CONCLUSION: The prevalence of C. difficile in children with diarrhoea was high (37.8%) although the proportion of toxigenic strains was comparatively low. The clinical significance of any isolate needs to be determined.

Ribosome Elongation Kinetics of Consecutively Charged Residues Are Coupled to Electrostatic Force.

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.

Impact of mechanical circulatory support on pediatric heart transplant candidates with elevated pulmonary vascular resistance.

With the new era of increasing use of mechanical circulatory support (MCS) in children, seemingly more patients with elevated pulmonary vascular resistance (PVR) are having positive outcomes. The purpose of this study was to define the effect of MCS on pediatric patients listed for heart transplant with an elevated PVR. The United Network for Organ Sharing (UNOS) database was used to identify patients aged 0-18 at the time of listing for heart transplant between 2010 and 2019 who had PVR documented (n = 2081). Patients were divided into MCS (LVAD, RVAD, BiVAD, and TAH) and No MCS groups, then divided by PVR (PVR) at the time of listing: <3, 3-6, and >6 Wood units (WU). MCS was used in 20% overall (n = 426); 57% of those with PVR <3, 27% with PVR 3-6, and 16% with PVR >6. MCS, PVR <3 patients had a higher chance of positive waitlist outcome than all No MCS groups (vs. PVR <3, P = .049; vs. PVR 3-6, P = .004; vs. PVR >6, P < .001). MCS, PVR 3-6 patients had a higher chance of positive waitlist outcome than all No MCS groups (vs. PVR <3, P = .048; vs. PVR 3-6, P = .009; vs. PVR >6, P < .001). MCS, PVR >6 patients had a higher chance of positive waitlist outcome than No MCS, PVR >6 patients (P = .012). Within the No MCS group, patients with a PVR >6 had a higher incidence of negative waitlist outcome compared to PVR <3 (17% vs. 10%, P = .002); this was not the case in the MCS group (5% vs. 6%, P = .693). More patients in the MCS group were ventilator dependent (15% vs. 9%, P < .001) at the time of listing and less likely to have a functional status >50% (43% vs. 73%, P < .001). No significant differences in post-transplant survival were found in pairwise comparisons of MCS and No MCS PVR subgroups. Patients supported with MCS had a significantly higher chance of a positive waitlist outcome than those without such support regardless of PVR status. This was most pronounced with a PVR greater than 6 WU. MCS compared to No MCS patients had better waitlist survival and equivalent post-transplant survival. MCS patients, despite being more ill, had better overall survival regardless of PVR.

Electrostatic Interactions Govern Extreme Nascent Protein Ejection Times from Ribosomes and Can Delay Ribosome Recycling.

The ejection of nascent proteins out of the ribosome exit tunnel, after their covalent bond to transfer-RNA has been broken, has not been experimentally studied due to challenges in sample preparation. Here, we investigate this process using a combination of multiscale modeling, ribosome profiling, and gene ontology analyses. Simulating the ejection of a representative set of 122 E. coli proteins we find a greater than 1000-fold variation in ejection times. Nascent proteins enriched in negatively charged residues near their C-terminus eject the fastest, while nascent chains enriched in positively charged residues tend to eject much more slowly. More work is required to pull slowly ejecting proteins out of the exit tunnel than quickly ejecting proteins, according to all-atom simulations. An energetic decomposition reveals, for slowly ejecting proteins, that this is due to the strong attractive electrostatic interactions between the nascent chain and the negatively charged ribosomal-RNA lining the exit tunnel, and for quickly ejecting proteins, it is due to their repulsive electrostatic interactions with the exit tunnel. Ribosome profiling data from E. coli reveals that the presence of slowly ejecting sequences correlates with ribosomes spending more time at stop codons, indicating that the ejection process might delay ribosome recycling. Proteins that have the highest positive charge density at their C-terminus are overwhelmingly ribosomal proteins, suggesting the possibility that this sequence feature may aid in the cotranslational assembly of ribosomes by delaying the release of nascent ribosomal proteins into the cytosol. Thus, nascent chain ejection times from the ribosome can vary greatly between proteins due to differential electrostatic interactions, can influence ribosome recycling, and could be particularly relevant to the synthesis and cotranslational behavior of some proteins.

Probing the Degradation Chemistry and Enhanced Stability of 2D Organolead Halide Perovskites.

Recent work on quasi-2D Ruddlesden-Popper phase organolead halide perovskites has shown that they possess many interesting optical and physical properties. Most notably, they are significantly more stable when exposed to moisture when compared to the typical 3D perovskite methylammonium lead iodide (MAPI); direct evidence for the chemical source of this stability remains elusive, however. Here, we present a detailed study of the superior moisture stability of a quasi-2D Ruddlesden-Popper perovskite, n-butylammonium methylammonium lead iodide (nBA-MAPI), compared to that of MAPI, and examine a simple, yet efficient, methodology to improve the stability of MAPI devices through the application of a thin layer of nBA-MAPI to the surface. By employing a variety of analytical techniques (photoluminescence, time-of-flight secondary ion mass spectrometry, cyclic voltammetry, X-ray diffraction) we determine that the improved stability of Ruddlesden-Popper perovskites is a consequence of a unique degradation pathway which produces a passivating surface layer, composed of increasingly stable phases of the 2D perovskite, via disproportionation. Our work establishes that this protective material isolates the bulk of the perovskite from a newly identified hydration layer which is found to accumulate at the C60/perovskite interface of full devices, slowing further hydrolysis reactions that would damage the device. As MAPI devices degrade quickly without any protection, a surface treatment of nBA-MAPI is an efficient way to delay device deterioration by creating an artificial 2D surface layer that similarly inhibits interaction with the hydration layer.

Cryptic Lineages and a Population Dammed to Incipient Extinction? Insights into the Genetic Structure of a Mekong River Catfish.

An understanding of the genetic composition of populations across management boundaries is vital to developing successful strategies for sustaining biodiversity and food resources. This is especially important in ecosystems where habitat fragmentation has altered baseline patterns of gene flow, dividing natural populations into smaller subpopulations and increasing potential loss of genetic variation through genetic drift. River systems can be highly fragmented by dams built for flow regulation and hydropower. We used reduced-representation sequencing to examine genomic patterns in an exploited catfish, Hemibagrus spilopterus, in a hotspot of biodiversity and hydropower development-the Mekong River basin. Our results revealed the presence of 2 highly divergent coexisting genetic lineages which may be cryptic species. Within the lineage with the greatest sample sizes, pairwise FST values, principal component analysis, and a STRUCTURE analysis all suggest that long-distance migration is not common across the Lower Mekong Basin, even in areas where flood-pulse hydrology has limited genetic divergence. In tributaries, effective population size estimates were at least an order of magnitude lower than in the Mekong mainstream indicating these populations may be more vulnerable to perturbations such as human-induced fragmentation. Fish isolated upstream of several dams in one tributary exhibited particularly low genetic diversity, high amounts of relatedness, and a level of inbreeding (GIS = 0.51) that has been associated with inbreeding depression in other outcrossing species. Our results highlight the importance of assessing genetic structure and diversity in riverine fisheries populations across proposed dam development sites for the preservation of these critically important resources.

Chemical Composition and Biological Activities of Metabolites from the Marine Fungi Penicillium sp. Isolated from Sediments of Co To Island, Vietnam.

Marine microorganisms are an invaluable source of novel active secondary metabolites possessing various biological activities. In this study, the extraction and isolation of the marine sediment Penicillium species collected in Vietnam yielded ten secondary metabolites, including sporogen AO-1 (1), 3-indolecarbaldehyde (2), 2-[(5-methyl-1,4-dioxan-2-yl)methoxy]ethanol (3), 2-[(2R-hydroxypropanoyl)amino]benzamide (4), 4-hydroxybenzandehyde (5), chrysogine (6), 3-acetyl-4-hydroxycinnoline (7), acid 1H-indole-3-acetic (8), cyclo (Tyr-Trp) (9), and 2',3'-dihydrosorbicillin (10). Their structures were identified by the analysis of 1D and 2D NMR data. Among the isolated compounds, 2-[(5-methyl-1,4-dioxan-2-yl)methoxy]ethanol (3) showed a strong inhibitory effect against Enterococcus faecalis with a minimum inhibitory concentration value of 32 µg/mL. Both 2-[(2R-hydroxypropanoyl)amino]benzamide (4) and 4-hydroxybenzandehyde (5) selectively inhibited E. coli with minimum inhibitory concentration values of 16 and 8 µg/mL, respectively. 2',3'-Dihydrosorbicillin (10) potentially inhibited α-glucosidase activity at a concentration of 2.0 mM (66.31%).

Effect of raw milk on lactose intolerance: a randomized controlled pilot study.

PURPOSE: This pilot study aimed to determine whether raw milk reduces lactose malabsorption and/or lactose intolerance symptoms relative to pasteurized milk. METHODS: We performed a crossover trial involving 16 adults with self-reported lactose intolerance and lactose malabsorption confirmed by hydrogen (H2) breath testing. Participants underwent 3, 8-day milk phases (raw vs 2 controls: pasteurized, soy) in randomized order separated by 1-week washout periods. On days 1 and 8 of each phase, milk consumption was 473 mL (16 oz); on days 2 to 7, milk dosage increased daily by 118 mL (4 oz), beginning with 118 mL (4 oz) on day 2 and reaching 710 mL (24 oz) on day 7. Outcomes were area under the breath H2 curve (AUC H2) and self-reported symptom severity (visual analog scales: flatulence/gas, audible bowel sounds, abdominal cramping, diarrhea). RESULTS: AUC H2 (mean ± standard error of the mean) was higher for raw vs pasteurized on day 1 (113 ± 21 vs 71 ± 12 ppm·min·10(-2), respectively, P = .01) but not day 8 (72 ± 14 vs 74 ± 15 ppm·min·10(-2), respectively, P = .9). Symptom severities were not different for raw vs pasteurized on day 7 with the highest dosage (P >.7). AUC H2 and symptom severities were higher for both dairy milks compared with soy milk. CONCLUSIONS: Raw milk failed to reduce lactose malabsorption or lactose intolerance symptoms compared with pasteurized milk among adults positive for lactose malabsorption. These results do not support widespread anecdotal claims that raw milk reduces the symptoms of lactose intolerance.

Synonymous Mutations Can Alter Protein Dimerization Through Localized Interface Misfolding Involving Self-entanglements.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

Recombinant structures expand and contract inter and intragenic diversification at the KIR locus.

BACKGROUND: The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total. RESULTS: Towards that end, we developed a KIR haplotyping method that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available. CONCLUSIONS: These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity of potential new KIR haplotypes derived from gene duplication, deletion, and hybridization.

Is Posttranslational Folding More Efficient Than Refolding from a Denatured State: A Computational Study.

The folding of proteins into their native conformation is a complex process that has been extensively studied over the past half-century. The ribosome, the molecular machine responsible for protein synthesis, is known to interact with nascent proteins, adding further complexity to the protein folding landscape. Consequently, it is unclear whether the folding pathways of proteins are conserved on and off the ribosome. The main question remains: to what extent does the ribosome help proteins fold? To address this question, we used coarse-grained molecular dynamics simulations to compare the mechanisms by which the proteins dihydrofolate reductase, type III chloramphenicol acetyltransferase, and d-alanine-d-alanine ligase B fold during and after vectorial synthesis on the ribosome to folding from the full-length unfolded state in bulk solution. Our results reveal that the influence of the ribosome on protein folding mechanisms varies depending on the size and complexity of the protein. Specifically, for a small protein with a simple fold, the ribosome facilitates efficient folding by helping the nascent protein avoid misfolded conformations. However, for larger and more complex proteins, the ribosome does not promote folding and may contribute to the formation of intermediate misfolded states cotranslationally. These misfolded states persist posttranslationally and do not convert to the native state during the 6 µs runtime of our coarse-grain simulations. Overall, our study highlights the complex interplay between the ribosome and protein folding and provides insight into the mechanisms of protein folding on and off the ribosome.

Fatty Acid Synthase Inhibitors Enhance Microtubule-Stabilizing and Microtubule-Destabilizing Drugs in Taxane-Resistant Prostate Cancer Cells.

Prostate cancer is the third leading cause of cancer-related death in men in the United States. Taxane chemotherapy is a staple therapy for men with metastatic prostate cancer, yet the median survival is less than 2 years in this setting. New strategies are needed to overcome taxane resistance to improve patient survival. Fatty acid synthase (FASN) is overexpressed in many types of cancer, and several inhibitors have been designed in the past 30 years. Previously, we showed that the FASN inhibitor orlistat was able to synergize with taxanes in two established taxane-resistant (TxR) cell lines. In the current study, we investigated five FASN inhibitors-cerulenin, orlistat, triclosan, thiophenopyrimidine fasnall, and pyrazole derivative TVB-3166 for their potential to synergize with docetaxel (a microtubule stabilizer) and vinblastine (a microtubule destabilizer) in TxR cell lines. Orlistat, TVB-3166, and fasnall synergistically inhibited cell viability when combined with docetaxel and vinblastine in PC3-TxR and DU145-TxR cells. Confocal microscopy and immunoblot with an antidetyrosinated tubulin antibody demonstrated that enhanced microtubule stability was induced by the combined treatment of FASN inhibitors and docetaxel compared with docetaxel alone, while combinations of FASN inhibitors with vinblastine diminished microtubule stability compared to vinblastine alone.

How soluble misfolded proteins bypass chaperones at the molecular level.

Subpopulations of soluble, misfolded proteins can bypass chaperones within cells. The extent of this phenomenon and how it happens at the molecular level are unknown. Through a meta-analysis of the experimental literature we find that in all quantitative protein refolding studies there is always a subpopulation of soluble but misfolded protein that does not fold in the presence of one or more chaperones, and can take days or longer to do so. Thus, some misfolded subpopulations commonly bypass chaperones. Using multi-scale simulation models we observe that the misfolded structures that bypass various chaperones can do so because their structures are highly native like, leading to a situation where chaperones do not distinguish between the folded and near-native-misfolded states. More broadly, these results provide a mechanism by which long-time scale changes in protein structure and function can persist in cells because some misfolded states can bypass components of the proteostasis machinery.

The driving force for co-translational protein folding is weaker in the ribosome vestibule due to greater water ordering.

Interactions between the ribosome and nascent chain can destabilize folded domains in the ribosome exit tunnel's vestibule, the last 3 nm of the exit tunnel where tertiary folding can occur. Here, we test if a contribution to this destabilization is a weakening of hydrophobic association, the driving force for protein folding. Using all-atom molecular dynamics simulations, we calculate the potential-of-mean force between two methane molecules along the center line of the ribosome exit tunnel and in bulk solution. Associated methanes, we find, are half as stable in the ribosome's vestibule as compared to bulk solution, demonstrating that the hydrophobic effect is weakened by the presence of the ribosome. This decreased stability arises from a decrease in the amount of water entropy gained upon the association of the methanes. And this decreased entropy gain originates from water molecules being more ordered in the vestibule as compared to bulk solution. Therefore, the hydrophobic effect is weaker in the vestibule because waters released from the first solvation shell of methanes upon association do not gain as much entropy in the vestibule as they do upon release in bulk solution. These findings mean that nascent proteins pass through a ribosome vestibule environment that can destabilize folded structures, which has the potential to influence co-translational protein folding pathways, energetics, and kinetics.

Characterization of envelope sequence of HIV virus in children infected with HIV in Vietnam.

BACKGROUND: HIV is characterized by high levels of genetic variability, including increased numbers of heterogeneous sequences of the envelope region. Therefore, studying genetic variability of HIV in relation to viral replication might facilitate prognosis of disease progression. METHODS: The study was designed as cross-sectional; data and samples of participants collected and analyzed env genes were obtained from 23 children enrolled by Vietnam National Children's Hospital. RESULTS: Substantial mutations in the C2 region were found in patients with high levels of viral replication while changes in the C3 region were mostly found in patients with low viral load. In the V1 region, we found profound amino acid modifications in patients with low HIV viral loads in contrast to the V2 sequence, where we identified single point mutations in patients with increased HIV viral load. The V3 region was relatively homogeneous, while profound deletions in the V4 region were detected in patients with increased viral replication. CONCLUSION: Our results suggest that genetic variations in different regions of the HIV envelope sequence, including both conserved C2 and C3 and variable V1/V2 and V4 regions, might be involved in increased viral infectivity and replication capacity. Such knowledge might help improve prediction of HIV progress and treatment in patients.