The GATA transcriptional program dictates cell fate equilibrium to establish the maternal-fetal exchange interface and fetal development.

The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors, and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of transcription factors GATA2 and GATA3 in labyrinth trophoblast progenitors (LaTPs) have underdeveloped placenta and die by ~embryonic day 9.5. Single-cell RNA sequencing analysis revealed excessive accumulation of multipotent LaTPs upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate into matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promotes trophoblast progenitor-to-SynT commitment, ensuring establishment of the maternal-fetal exchange interface.

Evaluation of Cassia fistula seed galactomannan as tablet-binder in formulation of diclofenac sodium-loaded monolithic matrix tablet.

The present study aimed to evaluate Cassia fistula seed galactomannan (CFSG) as a tablet-binder in the formulation of a monolithic matrix tablet using diclofenac sodium as a model drug. Initially, CFSG was extracted and purified from the seeds of the Cassia fistula tree and then screened for phytochemicals. Native CFSG was characterized with polysaccharide content determination, monosaccharide composition analysis, elemental analysis, FTIR, solid-state 13C NMR, molecular weight, zeta potential, DSC, TGA-DTA, XRD, viscosity, pH and surface tension, rheology, SEM and acute oral toxicity study. Prior to formulation, the drug-CFSG compatibility was checked by FTIR, DSC, and XRD. Diclofenac sodium-loaded granules were prepared by the wet granulation method and evaluated for various granule properties. Finally, granules were compressed into tablets and evaluated for binding and other tablet properties. The granules showed to have optimum micromeritic properties. Tablet hardness and friability were found to be approximately 7 kg/m2 and 0.3 %, respectively, which substantiate the excellent binding capacity of CFSG. Other tablet properties were also found to be within the Pharmacopoeial compliance limit. The tablets with a minimum concentration of CFSG (2.5%w/w) as binder showed appreciable mechanical strength and faster drug release, which ratifies CFSG as an alternative tablet binder.

The Role of C-reactive Protein Estimation in Determining the Duration of Antibiotic Therapy in Neonatal Sepsis.

INTRODUCTION: Septicemia is globally considered the most important cause of neonatal morbidity and fatality. Serum C-Reactive Protein (CRP) is an acute phase reactant, which is brought out in response to the inflammatory reaction. It is prophesied to drop down speedily after the coherent weeding out of microbial incitation due to the short half-life of CRP. CRP levels reflect the individual's association between microbial infection and defensive mechanisms.  Methods: This hospital-based cross-sectional study included 150 admitted patients with suspected sepsis in the Department of Pediatrics, Rajendra Institute Medical Sciences (RIMS), Ranchi, India, over a study period of one year (2020 to 2021). CRP was estimated on the day of admission and repeated after 72 hours, on the fifth day, and on the seventh day for serial values of CRP, and the findings were compared by making three groups. Further, the research participants were designated to three different groups according to the CRP estimation levels. RESULTS: Out of the 150 assumed neonatal septicemia patients, antibiotics were paused in 42 neonates (28%) within 72 hours. In group 2, 8% of neonates' antibiotics were stopped in five days, and a total of 102 neonates (68%) could be discharged on the seventh day of antibiotic therapy as their CRPs became negative on the third day and seventh day consecutively, along with negative blood culture reports. In group 3, antibiotics of 48 neonates (32%) were continued beyond seven days. CONCLUSION: CRP has a skyscraping specificity and negative predictive values (NPV); thus, by estimating serial CRPs, the antibiotic therapy duration can be determined, which further helps determine the period of hospitalization.

To study the association between various levels of cord serum albumin (CSA) and significant neonatal hyperbilirubinemia requiring interventions like phototherapy or exchange transfusion.

Introduction: Hyperbilirubinemia is most common normal physiological phenomenon in neonates affecting almost one third of newborn.it may lead to neuro disability leading to deafness and cerebral palsy which can be prevented if detected and treated as soon as possible. Albumin is produced in seventh week of intrauterine life and it can be measured by cord blood and in this study we can establish serum albumin with neonatal hyperbilirubinemia and can be treated by phototherapy or exchange transfusion. Material and Method: The study consists of 55 randomly selected eligible term neonates delivered at Rajendra Institute of Medical sciences from March 2019 to August 2020. Conclusion: In this study, in term neonates, level of serum albumin in umbilical cord less than 2.8 g/dl has no correlation with occurrence significant hyperbilirubinemia, so a level <2.8 gm/dl of serum albumin in umbilical cord blood can be used as critical value indicator in triaging predict the risk of occurring of significant hyperbilirubinemia in term neonates.level >3.4 gm/dl is considered safe in neonates who are the candidates for early discharge in the absence of other risk factors.

Hippo signaling cofactor, WWTR1, at the crossroads of human trophoblast progenitor self-renewal and differentiation.

Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.

Clinical Characteristics of Children With SARS-CoV-2 Infection During the Third Wave of the Pandemic: Single Center Experience.

OBJECTIVE: To evaluate pulmonary functions in children with transfusion-dependent thalassemia, and its reversal (lung dysfunction) using intensive intravenous chelation with desferrioxamine (DFO) (4 weeks). METHODS: This descriptive study enrolled 77 children with transfusion-dependent thalassemia. Pulmonary function test (PFT) and iron load (serum ferritin (SF) and T2* MRI of heart and liver) were done. PFT included spirometry, total lung capacity (TLC) by helium dilution test and diffusion capacity by carbon monoxide (DLCO). Follow-up PFT was available for 13 children with moderate to severe lung dysfunction given intravenous DFO. RESULTS: 50 (68.8%) patients had lung dysfunction, most commonly diffusional impairment (48; 96%), and reduced TLC (11; 22%); and none had obstructive pattern. 9 (81.8%) patients with restrictive defect had moderate to severely deranged DLCO. PFT and T2* MRI values were inversely correlated with serum ferritin. Among 13 patients receiving intensive chelation for 4 weeks, significant improvement was noticed in forced expiratory volume in one minute/ forced vital capacity ratio (DFEV1/FVC) (P=0.009), DDLCO (P=0.006) and DSF (P=0.01). CONCLUSIONS: Pulmonary dysfunction is common in children with multi-transfused thalassemia, and routine screening by PFT needs to be part of the management guidelines.

Drought and Heat Stress in Cool-Season Food Legumes in Sub-Tropical Regions: Consequences, Adaptation, and Mitigation Strategies.

Drought and heat stress are two major abiotic stresses that challenge the sustainability of agriculture to a larger extend. The changing and unpredictable climate further aggravates the efforts made by researchers as well as farmers. The stresses during the terminal stage of cool-season food legumes may affect numerous physiological and biochemical reactions that may result in poor yield. The plants possess a good number of adaptative and avoiding mechanisms to sustain the adverse situation. The various agronomic and breeding approaches may help in stress-induced alteration. The physiological and biochemical response of crops to any adverse situation is very important to understand to develop mechanisms and approaches for tolerance in plants. Agronomic approaches like altering the planting time, seed priming, foliar application of various macro and micro nutrients, and the application of rhizobacteria may help in mitigating the adverse effect of heat and drought stress to some extent. Breeding approaches like trait-based selection, inheritance studies of marker-based selection, genetic approaches using the transcriptome and metabolome may further pave the way to select and develop crops with better heat and drought stress adaptation and mitigation.

Effective clearance of uremic toxins using functionalised silicon Nanoporous membranes.

In-house fabricated silicon nanoporous membranes (SNMs), functionalized for efficient clearance of uremic toxins, can lead to compact and portable dialysis systems. Efficacy of 15 nm thick SNMs, with average pore diameter of 8 nm, was tested for dialysis of two uremic toxins - urea and creatinine using custom made teflon apparatus of 2, 10 and 30 ml. The apparatus consisted of two reservoirs, with the cis containing the uremic fluid, and the trans containing the dialysate. Peristalsis was found to enhance the clearance rate by a factor of four as compared to unstirred condition. Functionalisation of the SNMs reduced protein binding, and surface binding of urea from 23% to negligible values. A lateral array of nine SNMs and a new design for the dialysis apparatus, increased the clearance rate by a factor of twelve from that of the single SNM. The arrays cleared about 42% of urea and 48% of creatinine from 30 ml of diluted serum samples, in 15 min. Periodic replacement of the trans fluid cleared about 81% of high concentration uremic toxins from the cis reservoir in 45 mins. The SNM arrays are stable, reproducible, and with the superior clearance rates for urea and creatinine, they have the potential to be used as membranes for portable hemodialysers.

Surface adsorption of nitrosourea on pristine and doped (Al, Ga and In) boron nitride nanosheets as anticancer drug carriers: the DFT and COSMO insights.

To minimize the side effects of chemotherapeutic drugs and enhance the effectiveness of cancer treatment, it is necessary to find a suitable drug delivery carrier for anticancer drugs. Recently nanomaterials are extensively being studied as drug vehicles and transport drugs in tumor cells. Using DFT calculations, the adsorption behavior with electronic sensitivity and reactivity of pristine and doped (Al, Ga and In)-BNNS towards the nitrosourea (NU) drug has been investigated in gas as well as water media. Our calculations showed that the NU drug is physically adsorbed on the pristine BNNS with -0.49 and -0.26 eV by transferring little amount of charge of about 0.033e and 0.046e in gas and water media in the most stable complex. But after replacing one of the central B atoms with an Al or Ga or In atom, the sensitivity of the doped BNNS remarkably enhances towards the NU drug molecules. The NU drug prefers to be chemically adsorbed on the BN(Al)NS, BN(Ga)NS and BN(In)NS by -1.28, -1.58 and -3.06 eV in the gas phase and -1.34, -1.23 and -3.65 eV in water media in the most stable complexes respectively. The large destabilization of LUMO energies after the adsorption of the NU drug on the BN(Al)NS, BN(Ga)NS and BN(In)NS significantly reduces their E g from 4.37 to 0.69, 4.37 to 1.04 and 4.33 to 0.66 eV in the S1 complex respectively. The reduction of E g of doped BNNS by the NU drug greatly enhances the electrical conductivity which can be converted to an electrical signal. Therefore, this doped BNNS can be used as a fascinating electronic sensor for the detection of NU drug molecules. Furthermore the work function of the doped BNNS was largely affected by the NU drug adsorption about 47.3%, 39.3% and 40.4% in the gas phase and 41.3%, 36.6% and 31.6% in water media in the S1 complex of NU/BN(Al)NS, NU/BN(Ga)NS and NU/BN(In)NS respectively. Thus, the doped BNNS may be used as a Ф type sensor for NU drug molecules.

TEAD4 ensures postimplantation development by promoting trophoblast self-renewal: An implication in early human pregnancy loss.

Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.

Quality-by-design approach for development of sustained-release multiple-unit beads of lamotrigine based on ion-cross-linked composite of pectin and okra mucilage: An in vitro appraisal.

The main objective of the present study was to develop a sustained release multiple-unit beads of lamotrigine based on ionotropically cross-linked natural polysaccharides such as pectin (PTN) and okra mucilage (OM) and optimize the polymer-concentration, polymer ratio and cross-linker concentration by 23 full factorial design. Two different levels of three independent variables (total polymer concentration, polymer ratio and [CaCl2]) were considered for the experimental design. Drug-polymers compatibility was examined by FTIR, DSC, TGA and powder-XRD. The surface morphology of the bead before and after dissolution test was examined by SEM. Effects of the independent variables on bead-size, drug-encapsulation-efficiency (DEE), drug-release along with release similarity and difference factors were examined. The independent variables were then numerically optimized using Design-Expert software (Version 12) with the targets to meet USP-reference release profile after the analysis of variance of all the response parameters such as DEE, percent drug release at 2 h, 5 h, 12 h, Korsmeyer-Peppas rate constant, release similarity and difference factors. The optimized formulation showed excellent DEE of 89.2 ± 4.4% and a sustained release profile with release similarity factor of 94.9. Kinetic modeling of drug release data demonstrated a release mechanism combined of hydration, diffusion and erosion.

Atypical protein kinase C iota (PKCλ/ι) ensures mammalian development by establishing the maternal-fetal exchange interface.

In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.

Synthesis of MgO micro-rods coated with charred dextrose and its application for the adsorption of selected heavy metals from synthetic and real groundwater.

MgO micro-rods supported on porous carbon were synthesized by an economical method and applied for the adsorption of three different heavy metals ions (As (III), Cd (II) and Pb (II)). Here, we used dextrose as the source of carbon during the synthesis. The synthesized material has been characterized by different techniques like XRD, TEM, FE-SEM, BET and FT-IR for the determination of various physical properties. Compared with MgO synthesized without dextrose, the carbon-supported MgO or C-MgO demonstrated consistent rod-shaped morphology, higher surface area and better absorptivity. The adsorption data were analysed using various isotherm models and the Freundlich isotherm model seemed to provide the best fit to the data. The adsorption kinetics data on the other hand was well explicated by the pseudo second-order kinetic model. The maximum adsorption capacity of C-MgO was 508.47 mg g-1 for As (III), 566.01 mg g-1 for Cd (II) and 476.19 mg g-1 for Pb (II), respectively after 6 h of reaction. To check the real-life usability and efficiency of C-MgO, it was added to a groundwater sample which had 169.55 ppb of As (III) and within 20 min it was adsorbed with 99% efficiency. Reusability studies reveal that C-MgO could be used up to 6 times with more than 60% efficiency. This study shows that C-MgO has high adsorptive ability, is an economic and non-toxic material with versatile applications and can be used for groundwater remediation in real life.

A Novel Seed-Dressing Formulation Based on an Improved Mutant Strain of Trichoderma virens, and Its Field Evaluation.

Using gamma-ray-induced mutagenesis, we have developed a mutant (named G2) of Trichoderma virens that produced two- to three-fold excesses of secondary metabolites, including viridin, viridiol, and some yet-to-be identified compounds. Consequently, this mutant had improved antibiosis against the oomycete test pathogen Pythium aphanidermatum. A transcriptome analysis of the mutant vis-à-vis the wild-type strain showed upregulation of several secondary-metabolism-related genes. In addition, many genes predicted to be involved in mycoparasitism and plant interactions were also upregulated. We used tamarind seeds as a mass multiplication medium in solid-state fermentation and, using talcum powder as a carrier, developed a novel seed dressing formulation. A comparative evaluation of the wild type and the mutant in greenhouse under high disease pressure (using the test pathogen Sclerotium rolfsii) revealed superiority of the mutant over wild type in protecting chickpea (Cicer arietinum) seeds and seedlings from infection. We then undertook extensive field evaluation (replicated micro-plot trials, on-farm demonstration trials, and large-scale trials in farmers' fields) of our mutant-based formulation (named TrichoBARC) for management of collar rot (S. rolfsii) in chickpea and lentil (Lens culinaris) over multiple locations in India. In certain experiments, other available formulations were included for comparison. This formulation consistently, over multiple locations and years, improved seed germination, reduced seedling mortality, and improved plant growth and yield. We also noticed growth promotion, improved pod bearing, and early flowering (7-10 days) in TrichoBARC-treated chickpea and lentil plants under field conditions. In toxicological studies in animal models, this formulation exhibited no toxicity to mammals, birds, or fish.

Glycerol-mediated synthesis of nanoscale zerovalent iron and its application for the simultaneous reduction of nitrate and alachlor.

NZVI has long been used for the remediation of different groundwater contaminants but their tendency to get oxidized easily has always been a barrier to their reductive ability. In this work, we have made an attempt to enhance the aerobic stability of the nanoparticles by synthesizing them in a medium consisting of a viscous solvent, glycerol, and water. The XRD analysis of the nanoparticles reveals that the particles prepared in the presence of glycerol have a very thin coating of iron oxides on the outer surface of the nanoparticles in comparison with those prepared in the aqueous medium. These nanoparticles were applied for the simultaneous reduction of two groundwater contaminants, nitrate ions, and alachlor, which is an herbicide. Stock solutions of these two contaminants were prepared and then they were mixed in varying amounts and were treated by different doses of the nanoparticle. The optimized dose of the nanoparticles obtained for almost 97% removal of both the contaminants is 2.05 g/L. The studies showed that increasing the concentration of either of the contaminants while the other one was kept fixed led to a decrease in the removal efficiency. The studies conducted to see the effect of pH variation showed that the best removal can be achieved when the pH is 3 or even less than it, showing that acidic pH leads to higher removal values. Such nanoparticles which can be prepared easily at low-cost and can simultaneously act upon different contaminants are highly desired.

Facile one pot synthesis of zinc oxide nanorods and statistical evaluation for photocatalytic degradation of a diazo dye.

In the present work zinc oxide nanorods (ZNRs) have been synthesized to estimate its photocatalytic degradation potential on an industrially used diazo dye and optimization of the total treatment process has been designed. Response surface methodology (RSM) has been used to model the operational parameters for this photocatalytic degradation. The crystallite size (101 plane) of the synthesized ZNR has been found to be 20.99 nm having a band gap energy of 3.45 eV. At elevated pH, the rate of degradation of the photocatalyst was found to be higher than that of acidic pH. The independent variables of the model are time (9.6-122 min), pH (2-12.2), catalyst dose (0.2-0.4 g/L) and dye concentration (88-512 mg/L). It was seen that the degradation efficiency was significantly affected by the initial dye concentration and the pH, the optimal values of the parameters being a pH of 10.67, an initial concentration of 150 mg/L and ZnO dose of 0.37 g/L, the time taken being 88.52 min. The actual degradation efficiency of the dye reached 96.9% at optimized condition, which is quite close to the predicted value of 98.07%.

Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum.

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.

In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.

Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F.

Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.