Epithelial homelessness: an atypical form of anoikis triggered by Leishmania interaction with epithelial cells.

Aim: Leishmaniasis is characterized by a spectrum of diseases with two main clinical forms, cutaneous and visceral, caused by Leishmania tropica and Leishmania donovani, respectively. Studying Leishmania's interaction with the epithelial barrier at the initial site of a bite is crucial to understanding the establishment of the disease. Materials & methods: To discern parasite-host epithelial interaction, we developed in vitro cellular models involving co-cultures of Leishmania and MDCK epithelial cells. Results: Both L. donovani-MDCK and L. tropica-MDCK co-culture models demonstrated a phenomenon known as atypical anoikis apoptosis, typically identified by distinctive 'flipping in' of cell membranes and disordered cytoskeletal frameworks. Conclusion: This study bridges the gap in the fundamental understanding of the intricate latticework involving vector-Leishmania-host and may inform drug development strategies.

Small parasites called Leishmania are passed to humans through the bites of sandflies. These parasites cause three deadly forms of disease: one that affects the organs, one that causes skin lesions and one that affects organ linings. This study looked at how Leishmania parasites behave when they enter through the skin. We found that when the parasites were in contact with cells, the cells changed their shape and lost contact with neighboring cells. This led to a type of cell death known as anoikis, a Greek term meaning 'homelessness'.

Schizophrenia-associated NRXN1 deletions induce developmental-timing- and cell-type-specific vulnerabilities in human brain organoids.

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

Cuscuta reflexa Possess Potent Inhibitory Activity Against Human Malaria Parasite: An In Vitro and In Vivo Study.

Drug resistance to practically all antimalarial drugs in use necessitate the development of new chemotherapeutics against malaria. In this aspect, traditionally used plants with folklore reputation are the pillar for drug discovery. Cuscuta reflexa being traditionally used in the treatment of malaria in Odisha, India we aimed to experimentally validate its antimalarial potential. Different solvent extracts of C. reflexa or column fractions from a promising solvent extract were evaluated for in vitro anti-plasmodial activity against Plasmodium falciparum strain Pf3D7. Potent fractions were further evaluated for inhibition of parasite growth against different drug resistant strains. Safety of these fractions was determined by in vitro cyto-toxicity, and therapeutic effectiveness was evaluated by suppression of parasitemia and improvement in survival of experimental mice. Besides, their immunomodulatory effect was investigated in Pf-antigen stimulated RAW cells. GCMS fingerprints of active fractions was determined. Column separation of methanol extract which showed the highest in vitro antiplasmodial activity (IC50 = 14.48 µg/ml) resulted in eleven fractions, three of which (F2, F3, and F4) had anti-plasmodial IC50 ranging from ≤ 10 to 2.2 µg/ml against various P. falciparum strains with no demonstration of in vitro cytotoxicity. F4 displayed the highest in vivo parasite suppression, and had a mean survival time similar to artesunate (19.3 vs. 20.6 days). These fractions significantly modulated expression of inflammatory cytokines in Pf-antigen stimulated RAW cells. The findings of the study confirm the antimalarial potential of C. reflexa. Exploration of phyto-molecules in GCMS fingerprints of active fractions is warranted for possible identification of lead anti-malarial phyto-drugs.

Exposure to Propylparaben During Pregnancy and Lactation Induces Long-Term Alterations to the Mammary Gland in Mice.

The mammary gland is a hormone sensitive organ that is susceptible to endocrine-disrupting chemicals (EDCs) during the vulnerable periods of parous reorganization (ie, pregnancy, lactation, and involution). Pregnancy is believed to have long-term protective effects against breast cancer development; however, it is unknown if EDCs can alter this effect. We examined the long-term effects of propylparaben, a common preservative used in personal care products and foods, with estrogenic properties, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 20, 100, or 10 000 µg/kg/day propylparaben throughout pregnancy and lactation. Unexposed nulliparous females were also evaluated. Five weeks post-involution, mammary glands were collected and assessed for changes in histomorphology, hormone receptor expression, immune cell number, and gene expression. For several parameters of mammary gland morphology, propylparaben reduced the effects of parity. Propylparaben also increased proliferation, but not stem cell number, and induced modest alterations to expression of ERα-mediated genes. Finally, propylparaben altered the effect of parity on the number of several immune cell types in the mammary gland. These results suggest that propylparaben, at levels relevant to human exposure, can interfere with the effects of parity on the mouse mammary gland and induce long-term alterations to mammary gland structure. Future studies should address if propylparaben exposures negate the protective effects of pregnancy on mammary cancer development.

Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins.

Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosusl-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine, sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD polymerase (∼ 40 % increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications.

Stigmasterol as a potential biomarker for amphotericin B resistance in Leishmania donovani.

BACKGROUND: Leishmania donovani, a protozoan parasite, is the primary causative agent for visceral leishmaniasis. Toxicity and increased resistance to existing drugs have led to an urgent need for identifying new drugs and drug targets. Understanding the risks and mechanisms of resistance is of great importance. Amphotericin B (AmB) is a polyene antimicrobial, the mainstay therapy for visceral leishmaniasis in several parts of India. OBJECTIVES: In the present study, we established a line of AmB-resistant L. donovani promastigotes in vitro and demonstrated the molecular basis of resistance to AmB. METHODS: AmB-resistant promastigotes were generated and characterized to evaluate the mechanism of resistance to AmB. We examined the sterol composition of the promastigotes and the axenic amastigotes derived from the WT and AmB-resistant promastigotes. The role of the plant-like C-22 desaturase responsible for stigmasterol production was also evaluated in the AmB-resistant strain. RESULTS: The IC50 for resistant cells was four times higher than for the WT. AmB-resistant promastigotes showed an increase in the conversion of ß-sitosterol into stigmasterol. The presence of higher amounts of stigmasterol in resistant promastigotes, as well as in axenic amastigotes, signifies its role in AmB resistance in Leishmania. The resistant strain showed reduced infectivity in vitro. CONCLUSIONS: We have elucidated the mode of action and resistance mechanisms to the drug. However, further work is required to validate the potential role of stigmasterol in resistance and to help develop a diagnostic kit that can assist in diagnosing potentially resistant lines in the field.

CLARITY-BPA: Bisphenol A or Propylthiouracil on Thyroid Function and Effects in the Developing Male and Female Rat Brain.

The CLARITY-BPA experiment, a large collaboration between the National Institute of Environmental Health Sciences, the National Toxicology Program, and the US Food and Drug Administration, is designed to test the effects of bisphenol A (BPA) on a variety of endocrine systems and end points. The specific aim of this subproject was to test the effect of BPA exposure on thyroid functions and thyroid hormone action in the developing brain. Timed-pregnant National Center for Toxicological Research Sprague-Dawley rats (strain code 23) were dosed by gavage with vehicle control (0.3% carboxymethylcellulose) or one of five doses of BPA [2.5, 25, 250, 2500, or 25,000 µg/kg body weight (bw) per day] or ethinyl estradiol (EE) at 0.05 or 0.50 µg/kg bw/d (n = 8 for each group) beginning on gestational day 6. Beginning on postnatal day (PND) 1 (day of birth is PND 0), the pups were directly gavaged with the same dose of vehicle, BPA, or EE. We also obtained a group of animals treated with 3 ppm propylthiouracil in the drinking water and an equal number of concordant controls. Neither BPA nor EE affected serum thyroid hormones or thyroid hormone‒sensitive end points in the developing brain at PND 15. In contrast, propylthiouracil (PTU) reduced serum T4 to the expected degree (80% reduction) and elevated serum TSH. Few effects of PTU were observed in the male brain and none in the female brain. As a result, it is difficult to interpret the negative effects of BPA on the thyroid in this rat strain because the thyroid system appears to respond differently from that of other rat strains.

A Plant like Cytochrome P450 Subfamily CYP710C1 Gene in Leishmania donovani Encodes Sterol C-22 Desaturase and its Over-expression Leads to Resistance to Amphotericin B.

BACKGROUND: Leishmania donovani is a protozoan parasite, a primary causative agent of visceral leishmaniasis. Sterol produced via the mevalonate pathway, show differences in composition across biological kingdoms. The specific occurrence of Δ22-unsaturated sterols, containing a double bond at the C-22 position in the side chain occurs in fungi as ergosterol and as stigmasterol in plants. In the present study, we report the identification and functional characterization of a plant-like Cytochrome P450 subfamily CYP710C1 in L. donovani as the Leishmania C-22 desaturase. METHODOLOGY: In silico analysis predicted the presence of a plant like CYP710C1 gene that encodes a sterol C-22 desaturase, a key enzyme in stigmasterol biosynthesis. The enzymatic function of recombinant CYP710C1 as C-22 desaturase was determined. To further study the physiological role of CYP710C1 in Leishmania, we developed and characterized an overexpressing strain and a gene deletion mutant. C-22 desaturase activity and stigmasterol levels were estimated in the wild-type, overexpressing promastigotes and heterozygous mutants. CONCLUSION: We for the first time report the presence of a CYP710C1 gene that encodes a plant like sterol C-22 desaturase leading to stigmasterol biosynthesis in Leishmania. The recombinant CYP710C1 exhibited C-22 desaturase activity by converting ß-sitosterol to stigmasterol. Axenic amastigotes showed higher expression of CYP710C1 mRNA, protein and stigmasterol levels compared to the promastigotes. Sterol profiling of CYP710C1 overexpressing L. donovani and heterozygous mutant parasites demonstrated that CYP710C1 was responsible for stigmasterol production. Most importantly, we demonstrate that these CYP710C1 overexpressing promastigotes are resistant to amphotericin B, a drug of choice for use against leishmaniasis. We report that Leishmania sterol biosynthesis pathway has a chimeric organisation with characteristics of both plant and fungal pathways.

Comparative Analyses of the 12 Most Abundant PCB Congeners Detected in Human Maternal Serum for Activity at the Thyroid Hormone Receptor and Ryanodine Receptor.

Polychlorinated biphenyls (PCBs) pose significant risk to the developing human brain; however, mechanisms of PCB developmental neurotoxicity (DNT) remain controversial. Two widely posited mechanisms are tested here using PCBs identified in pregnant women in the MARBLES cohort who are at increased risk for having a child with a neurodevelopmental disorder (NDD). As determined by gas chromatography-triple quadruple mass spectrometry, the mean PCB level in maternal serum was 2.22 ng/mL. The 12 most abundant PCBs were tested singly and as a mixture mimicking the congener profile in maternal serum for activity at the thyroid hormone receptor (THR) and ryanodine receptor (RyR). Neither the mixture nor the individual congeners (2 fM to 2 µM) exhibited agonistic or antagonistic activity in a THR reporter cell line. However, as determined by equilibrium binding of [3H]ryanodine to RyR1-enriched microsomes, the mixture and the individual congeners (50 nM to 50 µM) increased RyR activity by 2.4-19.2-fold. 4-Hydroxy (OH) and 4-sulfate metabolites of PCBs 11 and 52 had no TH activity; but 4-OH PCB 52 had higher potency than the parent congener toward RyR. These data support evidence implicating RyRs as targets in environmentally triggered NDDs and suggest that PCB effects on the THR are not a predominant mechanism driving PCB DNT. These findings provide scientific rationale regarding a point of departure for quantitative risk assessment of PCB DNT, and identify in vitro assays for screening other environmental pollutants for DNT potential.

Oxybenzone Alters Mammary Gland Morphology in Mice Exposed During Pregnancy and Lactation.

Hormones and endocrine-disrupting chemicals are generally thought to have permanent "organizational" effects when exposures occur during development but not adulthood. Yet, an increasing number of studies have shown that pregnant females are disrupted by endocrine-disrupting chemical exposures, with some effects that are permanent. Here, we examined the long-term effects of exposure to oxybenzone, an estrogenic chemical found in sunscreen and personal care products, on the morphology of the mammary gland in mice exposed during pregnancy and lactation. Female mice were exposed to vehicle or 30, 212, or 3000 µg oxybenzone/kg/d, from pregnancy day 0 until weaning. A nulliparous group, receiving vehicle treatment, was also evaluated. Mammary glands were collected 5 weeks after involution for whole-mount, histological, immunohistochemical, and molecular analyses. Exposure to 3000 µg oxybenzone/kg/d induced permanent changes to ductal density that was significantly different from both the nulliparous and vehicle groups. The two highest doses of oxybenzone similarly induced an intermediate phenotype for expression of progesterone receptor. A monotonic, dose-dependent increase in cell proliferation was also observed in the oxybenzone-treated females, becoming statistically significant at the highest dose. Finally, oxybenzone exposure induced an intermediate phenotype for Esr1 expression in all oxybenzone-treated groups. These data suggest that oxybenzone, at doses relevant to human exposures, produces long-lasting alterations to mammary gland morphology and function. Further studies are needed to determine if exposure to this chemical during pregnancy and lactation will interfere with the known protection that pregnancy provides against breast cancer.

Hexadecylated linear PEI self-assembled nanostructures as efficient vectors for neuronal gene delivery.

Development of efficient and safe nucleic acid carriers is one of the most challenging requirements to improve the success of gene therapy. Here, we synthesized a linker, 3-(hexadecyloxy)-1-chloropropan-2-ol, and grafted it onto linear polyethylenimine in varying amounts to obtain a series of HD-lPEI polymers that were able to form self-assembled nanoparticles (SN). 1H-NMR spectrometry was used to determine the extent of grafting of the linker, HD, on to the lPEI backbone. We further complexed the SN of HD-lPEI with plasmid DNA (pDNA) and the resultant nanoplexes were characterized by their size and zeta potential and further evaluated for their transfection ability and cytotoxicity in MCF-7 cells. In the series, the SN of HD-lPEI-3 (ca. 15% substitution) showed the highest transfection efficiency (~ 91%) with non-significant cytotoxicity in comparison to the commercial transfection reagents. The in vitro gene knockdown study displayed ~ 80% suppression of GFP gene expression by SN of HD-lPEI-3/pDNA/siRNA complex, whereas Lipofectamine™/pDNA/siRNA complex could suppress the expression by only ~ 48%. The enhanced expression of luciferase gene using SN of HD-lPEI-3 in different vital organs of Balb/c mice also demonstrated the potential of the projected formulation for gene delivery. The encouraging results of SN of HD-lPEI-3 polymer for delivery of nucleic acids in vitro and in vivo paved the way to evaluate the potential of the same for neuronal siRNA delivery. The safe and efficient stereotaxic delivery of FITC-labeled siRNA against α-synuclein gene also confirms the potential applicability of HD-lEPI-3 SN as a vector for neuronal delivery.

Bisphenol S Alters the Lactating Mammary Gland and Nursing Behaviors in Mice Exposed During Pregnancy and Lactation.

High doses of estrogenic pharmaceuticals were once prescribed to women to halt lactation. Yet, the effects of low-level xenoestrogens on lactation remain poorly studied. We investigated the effects of bisphenol S (BPS), an estrogen receptor (ER) agonist, on the lactating mammary gland; the arcuate nucleus, a region of the hypothalamus important for neuroendocrine control of lactational behaviors; and nursing behavior in CD-1 mice. Female mice were exposed to vehicle, 2 or 200 µg BPS/kg/d from pregnancy day 9 until lactational day (LD) 20, and tissues were collected on LD21. Tissues were also collected from a second group at LD2. BPS exposure significantly reduced the fraction of the mammary gland comprised of lobules, the milk-producing units, on LD21, but not LD2. BPS also altered expression of Esr1 and ERα in the mammary gland at LD21, consistent with early involution. In the arcuate nucleus, no changes were observed in expression of signal transducer and activator of transcription 5, a marker of prolactin signaling, or ERα, suggesting that BPS may act directly on the mammary gland. However, observations of nursing behavior collected during the lactational period revealed stage-specific effects on both pup and maternal nursing behaviors; BPS-treated dams spent significantly more time nursing later in the lactational period, and BPS-treated pups were less likely to initiate nursing. Pup growth and development were also stunted. These data indicate that low doses of BPS can alter lactational behaviors and the maternal mammary gland. Together, they support the hypothesis that pregnancy and lactation are sensitive to low-dose xenoestrogen exposures.

Engineered polymeric amphiphiles self-assembling into nanostructures and acting as efficient gene and drug carriers.

Nonviral gene delivery systems are finding widespread use due to their safety, rapid and economical production, and ease of modification. In this work, series of N-alkyl-substituted linear polyethylenimine (CP) polymers have been synthesized, characterized, and investigated about how degree of substitution (hydrophobic-hydrophilic balance) (i.e. N-alkylation) influenced the transfection efficiency. Mobility shift assay demonstrated efficient binding of plasmid DNA (pDNA). Transfection efficiency and cytotoxicity of CP polymers were assessed in vitro, which revealed that all the formulations exhibited higher transfection activity than linear polyethylenimine (lPEI) and commercial transfection reagents, Lipofectamine and Superfect, with negligible toxicity (MTT assay). In the projected series, one of the formulations, CP-3-pDNA complex, displayed the highest transfection efficiency (∼1.6-12 folds vs. lPEI and commercial transfection reagents) and effectively carried GFP-specific siRNA inside the cells as monitored by measuring the suppression in the gene expression of the target gene. Further, flow cytometry experiments confirmed that CP-3-pDNA complex transfected the highest number of cells. Besides, CP-3 was also evaluated in terms of its capability to entrap hydrophobic drug molecules. The results showed that it efficiently encapsulated an anti-cancer drug, etoposide, and released it in a controlled fashion over a period of time. Altogether, the data support that CP-3 is a promising vector for nucleic acid as well as hydrophobic drug delivery.

Biodegradable and versatile polyethylenimine derivatives efficiently transfer DNA and siRNA into mammalian cells.

UNLABELLED: Polyethylenimines (PEIs) are considered as the most promising vectors for non-viral gene delivery applications. Here, we report the synthesis and in vitro evaluation of two non-toxic and biodegradable polymers, TEPA@bPEI (TBP) and TEPA@lPEI (TLP), derived from low molecular weight branched and linear polyethylenimines by the stepwise reactions with methylacrylate (aza-Michael reaction) and amidation with tetraethylenepentamine (TEPA). These polymers not only showed their ability to bind and condense pDNA into nano-sized complexes but also provided protection against nucleases in cellular milieu. Both the polymers exhibited excellent buffering capacity and efficiently delivered nucleic acids (plasmid DNA and siRNA) across the mammalian cells (CHO and A549 cells) and outclassed native polymers and the commercial transfection reagents in terms of transfection efficiency and target gene silencing, and that too without compromising on biocompatibility i.e. TOXICITY: The results advocate the promising potential of the PEI derivatives as safe and potent nucleic acid carriers for practical gene delivery applications.

Bioreducible polyethylenimine nanoparticles for the efficient delivery of nucleic acids.

Recently, non-viral vectors for nucleic acid delivery have received considerable attention. Among the various non-viral vectors, branched polyethylenimine (bPEI, 25 kDa) has been one of the most widely used carrier systems due to its high transfection efficiency, however, it imparts high cytotoxicity. In this study, we have crosslinked bPEI with a bioreducible linker, 3,3'-dithiodipropionic acid (DTPA), via electrostatic interactions to obtain DTPA crosslinked bPEI (DP) nanoparticles. The crosslinking significantly reduced the cytotoxicity of the nanoparticles. To arrive at the best formulation in terms of nucleic acid transfection, a series of DP nanoparticles were prepared by varying the percentage of crosslinking. The dual action of DTPA, i.e. partial blocking of the charge density as well as crosslinking to convert bPEI into its nanoparticles, did not alter the pDNA condensation ability of the so-formed nanoparticles, rather the strategy favoured the unpackaging of the complexes inside the cells improving the release of pDNA, which resulted in a higher transfection efficiency. All the formulations carried nucleic acids inside the cells and exhibited significantly higher transfection efficiencies than native bPEI and the commercial transfection reagent, Lipofectamine™. Sequential siRNA delivery displayed significant suppression in the target gene expression. All together, the evaluation of the delivery systems demonstrates that the newly synthesized DP NPs are quite promising as non-viral gene carriers.

Endocrine disruption in human placenta: expression of the dioxin-inducible enzyme, CYP1A1, is correlated with that of thyroid hormone-regulated genes.

CONTEXT: Thyroid hormone (TH) is essential for normal development; therefore, disruption of TH action by a number of industrial chemicals is critical to identify. Several chemicals including polychlorinated biphenyls are metabolized by the dioxin-inducible enzyme CYP1A1; some of their metabolites can interact with the TH receptor. In animals, this mechanism is reflected by a strong correlation between the expression of CYP1A1 mRNA and TH-regulated mRNAs. If this mechanism occurs in humans, we expect that CYP1A1 expression will be positively correlated with the expression of genes regulated by TH. OBJECTIVE: The objective of the study was to test the hypothesis that CYP1A1 mRNA expression is correlated with TH-regulated mRNAs in human placenta. METHODS: One hundred sixty-four placental samples from pregnancies with no thyroid disease were obtained from the GESTE study (Sherbrooke, Québec, Canada). Maternal and cord blood TH levels were measured at birth. The mRNA levels of CYP1A1 and placental TH receptor targets [placental lactogen (PL) and GH-V] were quantitated by quantitative PCR. RESULTS: CYP1A1 mRNA abundance varied 5-fold across 132 placental samples that had detectable CYP1A1 mRNA. CYP1A1 mRNA was positively correlated with PL (r = 0.64; P < .0001) and GH-V (P < .0001, r = 0.62) mRNA. PL and GH-V mRNA were correlated with each other (r = 0.95; P < .0001), suggesting a common activator. The mRNAs not regulated by TH were not correlated with CYP1A1 expression. CONCLUSIONS: CYP1A1 mRNA expression is strongly associated with the expression of TH-regulated target gene mRNAs in human placenta, consistent with the endocrine-disrupting action of metabolites produced by CYP1A1.

Polybrominated diphenyl ether (DE-71) interferes with thyroid hormone action independent of effects on circulating levels of thyroid hormone in male rats.

Polybrominated diphenyl ethers (PBDEs) are routinely found in human tissues including cord blood and breast milk. PBDEs may interfere with thyroid hormone (TH) during development, which could produce neurobehavioral deficits. An assumption in experimental and epidemiological studies is that PBDE effects on serum TH levels will reflect PBDE effects on TH action in tissues. To test whether this assumption is correct, we performed the following experiments. First, five concentrations of diphenyl ether (0-30 mg/kg) were fed daily to pregnant rats to postnatal day 21. PBDEs were measured in dam liver and heart to estimate internal dose. The results were compared with a separate study in which four concentrations of propylthiouracil (PTU; 0, 1, 2, and 3 ppm) was provided to pregnant rats in drinking water for the same duration as for diphenyl ether. PBDE exposure reduced serum T4 similar in magnitude to PTU, but serum TSH was not elevated by PBDE. PBDE treatment did not affect the expression of TH response genes in the liver or heart as did PTU treatment. PTU treatment reduced T4 in liver and heart, but PBDE treatment reduced T4 only in the heart. Tissue PBDEs were in the micrograms per gram lipid range, only slightly higher than observed in human fetal tissues. Thus, PBDE exposure reduces serum T4 but does not produce effects on tissues typical of low TH produced by PTU, demonstrating that the effects of chemical exposure on serum T4 levels may not always be a faithful proxy measure of chemical effects on the ability of thyroid hormone to regulate development and adult physiology.

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes.

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ~2.0-7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.

Copolymers of covalently crosslinked linear and branched polyethylenimines as efficient nucleic acid carriers.

The present study describes the formation of copolymers of linear and branched PEIs (25 kDa each). These polyethylenimines (bPEI and IPEI) were crosslinked with each other to obtain branched-linear (BL) PEI copolymers using epichlorohydrin as a crosslinker in two steps. First, IPEI was reacted with epichlorohydrin to form IPEI-chlorohydrin (CHL) and subsequently, bPEI was grafted onto CHL in basic medium by in situ generation of epoxy functionalities. The two PEIs were crosslinked by varying the weight ratio of bPEI while keeping the amount of IPEI fixed. The ratio of two PEIs (1:1, 2:1, 3:1, 4:1 and 5:1) and crosslinking percentage of epichlorohydrin (5, 10, 15 and 20%) appeared as the main parameters to have affected the transfection efficiency. The lead conjugate/DNA complex was tested for in vivo transgene expression in Balb/c mice and was found to show maximum expression in the spleen.

Oligoamine-tethered low generation polyamidoamine dendrimers as potential nucleic acid carriers.

In recent years, dendrimers have emerged as the most widely explored materials for theranostics emphasizing their potential in therapeutic delivery and diagnostics as well as in pharmaceutical technology. Amongst them, PAMAM dendrimers have been extensively studied for their prospects in various biomedical applications due to their defined structures and distinctive features such as monodispersity, uniformity and amenability to functionalization. Here, low generation PAMAM dendrimers (G2-G4) have been modified via Michael addition reaction followed by amidation with an oligoamine linker, tetraethylenepentamine (TEPA). Subsequently, these modified dendrimers were characterized by physicochemical techniques and evaluated for their capability to transfer nucleic acids in vitro. The results displayed significant improvements in the transfection efficiency in both HeLa and A549 cells maintaining higher cell viability. Sequential delivery of GFP-specific siRNA resulted in ∼73% suppression of the target gene. Flow cytometry results revealed that one of the formulations, mG3-pDNA complex, exhibited the highest gene transfection (∼49-68%) outperforming pDNA complexes of native dendrimers and the standard transfection reagent, Superfect (∼32-36%). All these results ensure the potential of the modified dendrimers as effective vectors for future gene delivery applications.