P1 Region Research Articles

Preclinical Metabolism and Disposition of an Orally Bioavailable Macrocyclic FXIa Inhibitor

FXIa-6f is as a high affinity, orally bioavailable macrocyclic FXIa inhibitor with antithrombotic activity in preclinical species. The objectives of this study were to characterize the in vitro metabolism, determine circulating metabolites in pre-clinical species, and examine the disposition of the compound in a bile duct-cannulated rat study (BDC) study, to inform clinical development of the compound and the medicinal chemistry approach to identify molecules with improved properties. Across species, metabolic pathways included several oxidative metabolites, including hydroxylated metabolites on the macrocycle or P1 region, descarbamoylation of the methyl carbamate side chain, and a glutathione conjugate on the 2,6-difluoro-3-chlorophenyl ring. In BDC rat, the absorbed dose of [3H]FXIa-6f was cleared mainly by metabolism, with excretion of drug-related material in the bile, mostly as metabolites. In all preclinical species, the parent drug was the primary drug-related component in circulation, but the species differences in the metabolic pathways observed in vitro were reflected in the plasma, where M6 was more prominent in rat plasma, and M9 was more prominent in monkey plasma. Based on the available data, the human metabolism appears to be most similar to monkey.

Immunovirological Assessment of HIV-Infected Patients and Phylogenetic Analysis of the Virus in Northeast of Iran

Background: The reliable laboratory tests, co-infection with other tumor viruses, and determining the genetic types are very important for therapy and monitoring of the clinical status of human immunodeficiency virus (HIV)-infected subjects. Objectives: This study evaluated the co-infection of HIV with hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type 1 (HTLV-1), the viral load, the progression of infection, and its correlation with the clinical status. Methods: Twenty HIV-infected cases were assessed for T cell subpopulations, HBV, HCV, and HTLV-1 co-infection, as well as the viral load. For phylogenetic relationships analysis, the HIV-c2-v5 fragment and p17 of gag were amplified, sequenced, and then clustered using phylogenetic analysis by MEGA software with maximum-likelihood. Results: The quantity of HIV viral load by qRT-PCR (TaqMan) and Cobas-Amplicor monitor test had a very strong correlation (R = 0.881, P < 0.0001). A significant negative correlation was also found between CD4+ cell count and Cobas-Amplicor (R = -0.41, P = 0.06). A significant negative correlation was also found between CD4+ cell count and Cobas-Amplicor (R = -0.41, P = 0.06) with HIV monitor test results (R = -0.41, P = 0.06). The phylogenetic analysis for p17 regions in gag and c2-v5 in env genes showed that all subjects had AD genotype. The co-infection of the HIV subjects with HBV, HCV, and HTLV-1 was 75%, 75%, and 15%, respectively. A direct correlation was observed between CD8+ and HIV-HTLV-1 co-infection. Conclusions: The results showed that HIV CRF35-AD, (M group) is more frequent in the northeast of Iran, and both real-time quantification methods were reliable for monitoring the HIV-1 viral load. In addition, the transmission rate of HTLV-1 is lower than HBV and HCV among drug abusers.

Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.

Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

GmPTF1 modifies root architecture responses to phosphate starvation primarily through regulating GmEXPB2 expression in soybean.

Though root architecture modifications may be critically important for improving phosphorus (P) efficiency in crops, the regulatory mechanisms triggering these changes remain unclear. In this study, we demonstrate that genotypic variation in GmEXPB2 expression is strongly correlated with root elongation and P acquisition efficiency, and enhancing its transcription significantly improves soybean yield in the field. Promoter deletion analysis was performed using 5' truncation fragments (P1-P6) of GmEXPB2 fused with the GUS gene in soybean transgenic hairy roots, which revealed that the P1 segment containing three E-box elements significantly enhances induction of gene expression in response to phosphate (Pi) starvation. Further experimentation demonstrated that GmPTF1, a basic-helix-loop-helix transcription factor, is the regulatory factor responsible for the induction of GmEXPB2 expression in response to Pi starvation. In short, Pi starvation induced expression of GmPTF1, with the GmPTF1 product directly binding to the E-box motif in the P1 region of the GmEXPB2 promoter. Plus, both GmPTF1 and GmEXPB2 highly expressed in lateral roots, and were significantly enhanced by P deficiency. Further work with soybean stable transgenic plants through RNA sequencing analysis showed that altering GmPTF1 expression significantly impacted the transcription of a series of cell wall genes, including GmEXPB2, and thereby affected root growth, biomass and P uptake. Taken together, this work identifies a novel regulatory factor, GmPTF1, involved in changing soybean root architecture partially through regulation of the expression of GmEXPB2 by binding the E-box motif in its promoter region.

Phylogenetic evidence of HIV-1 transmission linkage between two men who have sex with men

BackgroundIn China, an HIV-infected man (complainant; P2) alleged that another man (defendant; P1) had unlawfully infected him with HIV through unprotected homosexual contact in 2018.MethodsWe employed epidemiological, serological and phylogenetic analyses to investigate the transmission linkage between two men who have sex with men (MSM). Partial segments of three HIV-1 gene regions (gag, pol, and env) were amplified and sequenced by cloning. Maximum-likelihood (ML) and Bayesian methods were used to determine the direction and estimate the timing of transmission. Local control sequences and database control sequences were also used in the phylogenetic analysis.ResultsIt indicated that P2 underwent HIV seroconversion after P1 was diagnosed as HIV positive. The time to the most recent common ancestor (tMRCA) estimates consistently showed that P1 most likely became HIV-1 infected at an earlier date than P2. P1 and P2 were infected with the same HIV-1 CRF01_AE subtype according to segments of all three gene regions (gag, pol, and env). All three genetic regions of P1 have been subject to more potential selective forces than those of P2, indicating a longer evolutionary history. Bayesian and ML trees showed similar paraphyletic-monophyletic topologies of gag and env, with the virus from P1 located at the root, which supported a P1-to-P2 transmission direction.ConclusionsPhylogenetic investigations can elucidate HIV transmission linkage and might empower its use in the opposition of the intentional transmission of HIV-1 as a forensic tool.

HTERT Promotes CRC Proliferation and Migration by Recruiting YBX1 to Increase NRF2 Expression.

High human telomerase reverse transcriptase (hTERT) expression is related to severe Colorectal Cancer (CRC) progression and negatively related to CRC patient survival. Previous studies have revealed that hTERT can reduce cancer cellular reactive oxygen species (ROS) levels and accelerate cancer progression; however, the mechanism remains poorly understood. NFE2-related factor 2 (NRF2) is a molecule that plays a significant role in regulating cellular ROS homeostasis, but whether there is a correlation between hTERT and NRF2 remains unclear. Here, we showed that hTERT increases CRC proliferation and migration by inducing NRF2 upregulation. We further found that hTERT increases NRF2 expression at both the mRNA and protein levels. Our data also revealed that hTERT primarily upregulates NRF2 by increasing NRF2 promoter activity rather than by regulating NRF2 mRNA or protein stability. Using DNA pull-down/MS analysis, we found that hTERT can recruit YBX1 to upregulate NRF2 promoter activity. We also found that hTERT/YBX1 may localize to the P2 region of the NRF2 promoter. Taken together, our results demonstrate that hTERT facilitates CRC proliferation and migration by upregulating NRF2 expression through the recruitment of the transcription factor YBX1 to activate the NRF2 promoter. These results provide a new theoretical basis for CRC treatment.

Subtype-specific differences in Gag-protease replication capacity of HIV-1 isolates from East and West Africa

BackgroundThe HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower.ResultsGag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa.ConclusionsAlthough prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.

Role of the Proximal Region of CYP1A2 in the Homomeric P450 Complex Formation

Previous studies have established the formation of the CYP1A2-CYP1A2 homomeric complex and its effect on metabolic function. Preliminary evidence has shown that CYP1A2 activity exhibits a sigmoidal response in the CYP1A2/POR binary system as a function of POR. As the CYP1A2‑CYP1A2 homomeric complex is disrupted, CYP1A2 activity is increased, which is consistent with cooperativity. The goal of this study was to identify the regions of CYP1A2 involved in the homomeric complex formation. Structural evidence suggested that the proximal face of CYP1A2 may play a role in the homomeric complex formation. Consequently, a part of the proximal face (residues L91-K106: P1 region) was examined. Residues in this region of CYP1A2 were replaced with the homologous region of CYP2B4 (T81‑S96) using molecular cloning and was expressed in HEK-293T cells. Bioluminescence resonance energy transfer (BRET) was used to measure complex formation and disruption. The chimeric CYP1A2 (with the mutated P1 region) showed a decreased BRET signal when co‑transfected with wild-type CYP1A2, thereby indicating a decrease in complex formation. This was further verified by competition studies, which showed that the CYP1A2-P1 chimera was less effective at disrupting the CYP1A2 homomeric complexes as compared to wild‑type CYP1A2. This effectively demonstrated the impaired binding of the CYP1A2-P1 chimera to wild-type CYP1A2. Lower activity was observed as well with the CYP1A2‑P1 chimera as compared to wild‑type CYP1A2. As POR associates with CYP1A2 on the proximal face, the decreased activity of the chimera indicated that the ability of the chimera to interact with POR was affected. The chimera exhibited a decreased BRET signal when co‑transfected with wild‑type CYP1A2. Competition studies demonstrated that the chimera was less effective than wild‑type CYP1A2 at disrupting the CYP1A2‑POR heteromeric complex. Therefore, the binding of the chimera to POR was impaired, which was consistent with the decreased activity of the chimera. Therefore, these results not only establish the P1 region as a contact region in the CYP1A2‑CYP1A2 homomeric complex formation but also that modification of P1 region also affects formation of the CYP1A2‑POR heteromeric complex.

A Vaccine Strain of the A/ASIA/Sea-97 Lineage of Foot-and-Mouth Disease Virus with a Single Amino Acid Substitution in the P1 Region That Is Adapted to Suspension Culture Provides High Immunogenicity.

There are seven viral serotypes of foot-and-mouth disease virus (FMDV): A, O, C, Asia 1, and Southern African Territories 1, 2, and 3 (SAT 1–3). Unlike serotype O FMDV vaccine strains, vaccine strains of serotype A FMDV do not provide broad-range cross-reactivity in serological matching tests with field isolates. Therefore, the topotype/lineage vaccine strain circulating in many countries and a highly immunogenic strain might be advantageous to control serotype A FMDV. We developed a new vaccine strain, A/SKR/Yeoncheon/2017 (A-1), which belongs to the A/ASIA/Sea-97 lineage that frequently occurs in Asian countries. Using virus plaque purification, we selected a vaccine virus with high antigen productivity and the lowest numbers of P1 mutations among cell-adapted virus populations. The A/SKR/Yeoncheon/2017 (A-1) vaccine strain has a single amino acid mutation, VP2 E82K, in the P1 region, and it is perfectly adapted to suspension culture. The A/SKR/Yeoncheon/2017 (A-1) experimental vaccine conferred high immunogenicity in pigs. The vaccine strain was serologically matched with various field isolates in two-dimensional virus neutralization tests using bovine serum. Vaccinated mice were protected against an A/MAY/97 virus that was serologically mismatched with the vaccine strain. Thus, A/SKR/Yeoncheon/2017 (A-1) might be a promising vaccine candidate for protection against the emerging FMDV serotype A in Asia.

Isolation and genetic characteristics of a neurotropic teschovirus variant belonging to genotype 1 in northeast China.

Porcine teschovirus (PTV) is a causative agent of reproductive disorders, encephalomyelitis, respiratory diseases, and diarrhea in swine, with a worldwide distribution. In this work, we identified PTV-associated nonsuppurative encephalitis as a potential cause of posterior paralysis in neonatal pigs in northeast China. Using indirect immunofluorescence assay, western blot, electron microscopy, and genome sequencing, we identified a neurotropic PTV strain, named CHN-NP1-2016, in the supernatants of pooled cerebrum and cerebellum samples from an affected piglet. Nucleotide sequence alignment revealed that the whole genome of CHN-NP1-2016 shared the highest sequence similarity (86.76% identity) with PTV 1 strain Talfan. A combination of phylogenetic and genetic divergence analysis was applied based on the deduced amino acid sequence of the P1 gene with a cutoff value of the genetic distance (0.102 ± 0.008) for defining PTV genotypes, and this showed that CHN-NP1-2016 is a variant of genotype 1. In total, 16 unique mutations and five mutant clusters were detected in the capsid proteins VP1 and VP2 of CHN-NP1-2016 when compared to other PTV1 isolates. Importantly, we detected three mutant clusters located in the exposed surface loops of the capsid protein, potentially indicating significant differences in major neutralization epitopes. Moreover, a potential recombination event in the P1 region of PTV CHN-NP1-2016 was detected. These findings provide valuable insights into the role of recombination in the evolution of teschoviruses. To our knowledge, this is the first case report of PTV-1-associated encephalitis in northeast China. Future investigations will narrow on the serology and pathogenicity of this novel isolate.

The N-terminus of the cauliflower mosaic virus aphid transmission protein P2 is involved in transmission body formation and microtubule interaction

Cauliflower mosaic virus (CaMV) is transmitted by aphids using the non-circulative transmission mode: when the insects feed on infected leaves, virus particles from infected cells attach rapidly to their stylets and are transmitted to a new host when the aphids change plants. Mandatory for CaMV transmission, the viral helper protein P2 mediates as a molecular linker binding of the virus particles to the aphid stylets. P2 is available in infected plant cells in a viral inclusion that is specialized for transmission and named the transmission body (TB). When puncturing an infected leaf cell, the aphid triggers an ultra-rapid viral response, necessary for virus acquisition and called transmission activation: The TB disrupts and P2 is redistributed onto cortical microtubules, together with virus particles that are simultaneously set free from virus factories and join P2 on the microtubules to form the so-called mixed networks (MNs). The MNs are the predominant structure from which CaMV is acquired by aphids. However, the P2 domains involved in microtubule interaction are not known. To identify P2 regions involved in its functions, we generated a set of P2 mutants by alanine scanning and analyzed them in the viral context for their capacity to form a TB, to interact with microtubules and to transmit CaMV. Our results show that contrary to the previously characterized P2-P2 and P2-virion binding sites in its C-terminus, the microtubule binding site is contained in the N-terminal half of P2. Further, this region is important for TB formation since some P2 mutant proteins did not accumulate in TBs but were retained in the viral factories where P2 is translated. Taken together, the N-terminus of P2 is not only involved in vector interaction as previously reported, but also in interaction with microtubules and in formation of TBs.

Aerobic Exercise Mediates Neural Inefficiency Among Adolescents At Risk for Suicide.

This study examined the electroencephalograpy (EEG) coherence and working memory performance effects of a 9-week exercise program on a small group of adolescents at high suicide risk. We randomly assigned 26 adolescents at high suicide risk (based on their scores on the Adolescents Mental Health Inventory- AMHI) into equal sized exercise and no-exercise groups. Before and after the 9-week exercise program, all participants performed the Sternberg working memory task, during which we recorded their EEGs, with electrodes placed at F3, F4, C3, C4, P3, P4, T3, T4, O1, and O2 regions (using the International 10-20 system of EEG electrode placement). We measured working memory performance and inter-hemispheric (F3-F4, C3-C4, T3-T4, P3-P4, O1-O2) and intra-hemispheric (F3-C3, F3-T3, F3-P3, F3-O1, F4-C4, F4-T4, F4-P4, F4-O2) EEG coherence as an index of the participants' underlying neural efficiency. While we found no significant group differences in working memory performances, the exercise group, relative to the no-exercise group, exhibited lower inter- and intra-hemispheric EEG coherence while performing the working memory task. These EEG differences may reflect a mediating effect of aerobic exercise on these participants' neural efficiency.

Molecular epidemiology and recombination of Enterovirus A71 in mainland China from 1987 to 2017.

Enterovirus A71 (EV-A71) is an important pathogen of severe hand, foot, and mouth disease (HFMD) in young children. This study aimed to retrospectively analyze the molecular epidemiology and recombination of EV-A71 in mainland China during 1987–2017. Phylogenetic tree showed that besides the previously reported subgenotypes A, B5, C0, C2, C3, and C4, a new subgenotype C6 emerged in mainland China. Recombination analysis indicated that C4 EV-A71 was derived from a common ancestor as a “double-recombinant” virus by intertypic recombination between C EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains in P3 region and intratypic recombination between C and B EV-A71 strains in P2 region. The B5 EV-A71 shared high similarity with C EV-A71 in P1 region while it contained an unidentified sequence in P2 and P3 regions with two possible recombination patterns: one occurred between C4 EV-A71 and CVA3, CVA5, CVA6, CVA10, and CVA12 stains with one breakpoint in 3C, and the other occurred between C1, C2, C3, and C5 EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains with two breakpoints in the 2A/2B junction and 3C. The C2 EV-A71 was probably a recombinant virus between C4 EV-A71 and CVA8 strains with two breakpoints located in the 5′UTR and 2A/2B junction. Moreover, an incredible recombination of C6 EV-A71 occurred between C4 and C2 EV-A71 with multiple breakpoints. Thus, continuous studies on EV-A71 genome characteristics are still useful and essential for monitoring emergence of new viruses and preventing HFMD outbreaks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10123-021-00164-2.

Crystallization of a nonreplicating rotavirus vaccine candidate.

Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first‐principles models and preliminary experimental data. The first‐principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging‐drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled‐up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV.

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.

Molecular and genetic characterization of hepatitis B virus among multitransfused thalassaemia patients in Islamabad, Pakistan.

Background:Hepatitis B virus (HBV) is the aetiological agent of transfusion-transmitted hepatitis globally. Beta thalassaemia major individuals are at greater risk of contracting HBV infection due to multiple blood transfusions required for the medical management of these patients. Based on HBV genetic variability, it is divided into 10 genotypes. The determination of HBV genotypes has significant implications for clinical management and treatment regimens.Aim:This study was performed to assess the HBV epidemiology and circulating genotypes in multi-transfused β-thalassemia major patients with the aim to be considered while formulating the treatment pattern taking into account particular needs of thalassaemia patients.Materials and Methods:This study was performed from September 2018 to June 2019, at the Department of Pathology and Transfusion Medicine, Shaheed Zulfiqar Ali Bhutto (SZAB) Medical University, Islamabad. A total of 2,260 thalassaemia patients were enrolled in the study. The study was endorsed by the Ethics Committee of the SZAB Medical University, Islamabad. The samples were serologically screened for HBsAg on the LIAISON® XL Murex HBsAg Quant assay (DiaSorin S.p.A., Italy) a chemiluminescence based immunoassay (CLIA). HBV quantitative PCR kit was used to measure the HBV DNA in serum samples. The HBV genotypes were determined using universal primers targeting the P1 and S1 region amplification.Results:Of 2,260 thalassaemia patients, 64.6% were males while 35.4% were females. The HBsAg was identified in 98 individuals (4.33%). The PCR analysis was done for these 98 patients and in this cohort, genotype D was 59.18% (n = 58), genotype A was 21.42% (n = 21) while genotype C was 19.38% (n = 19).Conclusion:The determination of HBV genotypes in the multi-transfused patients is key to the effective management of chronic HBV patients as the severity and course of the disease is dependent on a specific type of genotypes. Quality assured screening of donated blood will prevent the incidence of HBV in thalassaemia patients.

Molecular level insights into the inhibition of gene expression by thiamine pyrophosphate (TPP) analogs for TPP riboswitch: A well-tempered metadynamics simulations study

Riboswitches are metabolite sensing aptamer domains present in non-coding regions in RNA and act as gene-regulating elements. Thiamine pyrophosphate (TPP) riboswitch is evolved as a new target for developing antibiotics against many pathogenic bacteria. The earlier reports suggest that the modification of the pyrophosphate group in the ligand molecule can enhance gene expression. In this work, we have examined the binding affinity and efficacy of TPP and two recently reported ligands, CH2-TPP, and CF2-TPP, using Well-tempered metadynamics (WT-MtD) simulations. The experimental in vitro assays show that both TPP and CH2-TPP repress the gene expression to the same extent. The calculated binding energies correlate well with the experimental study and show the same trend of binding affinity of ligands for the TPP riboswitch. The root mean square fluctuation profiles suggest that the CH2-TPP and TPP trigger higher fluctuations in P1 and L3 region, and such fluctuations in the P1 region is involved in the gene regulation process. The metal ion mediated contact of TPP ligand with pyrophosphate binding helix is found to be critical in the gene regulation process. The simulation results corroborate the experimental observations that the role of conformational changes occurring in different riboswitch regions upon ligand binding is essential to repress the gene expression process. This work sheds light on the subtle change in the ligand structure that can induce a more considerable impact on binding affinity and efficacy of ligands with riboswitch.

Use of Synonymous Deoptimization to Derive Modified Live Attenuated Strains of Foot and Mouth Disease Virus.

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that “synonymous codon deoptimization” could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.

Two Natural Recombination gave rise to the Coxsackievirus B3 GV that triggered outbreaks in China in 2006 - 2012

Coxsackievirus B3 serotype GV caused the epidemic of Coxsackievirus B3 infection in China from 2006 to 2012. To study the evolution and recombination of Coxsackievirus B3 serotype GV, we performed recombination and phylogenetic analysis of 499 complete genomes of Enterovirus B available in GenBank, dated April 2019. Results indicated that most of the strains of Coxsackievirus B3 GV in P1 region were derived from a Coxsackievirus B3 GVI parent, and in P2-3 region from EchoV E25 strain, with nucleotide identities of 97.2% and 94.7%, respectively. Other strains of Coxsackievirus B3 GV-C1 in P1-P2 regions were derived from Coxsackievirus B3 GV-C3, whereas those in P3 regions were from CVB5. These naturally occurring recombination events were confirmed by phylogenetic analysis. This study indicates that two naturally occurring recombination gave rise to the coxsackievirus B3 GV that triggered outbreaks in China in 2006 - 2012.

Cortical Activity During Postural Recovery in Response to Predictable and Unpredictable Perturbations in Healthy Young and Older Adults: A Quantitative EEG Assessment.

Introduction:To investigate the effects of predictable and unpredictable external perturbations on cortical activity in healthy young and older adults.Methods:Twenty healthy older and 19 healthy young adults were exposed to predictable and unpredictable external perturbations, and their cortical activity upon postural recovery was measured using a 32-channel quantitative encephalography. The absolute spectral power and coherence z-scores of cortical waves were analyzed through a 3-way mixed ANOVA.Results:During postural recovery from predictable perturbations, older adults exhibited higher frontoparietal beta power and higher alpha and beta coherence during the late-phase recovery than the young individuals. After unpredictable perturbations, the older group showed lower alpha power in the early phase and higher beta power in the late phase as compared to the young group. Results for the group × time and group × location interactions in the older group showed a higher alpha and beta coherence over the late phase, a higher alpha coherence in F3–P3 and F4–P4 regions, and a higher beta coherence in the F4–P4 region compared to the younger group.Conclusion:Our results revealed that the cortical activation after external perturbations increases with aging, particularly in frontoparietal areas. A shift from automatic (subcortical level) to attentional (cortical level) processing may reflect the contribution of attentional resources for postural recovery from an external threat in older individuals.