Factors affecting macromolecule orientations in thin films formed in cryo-EM.

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.

Assessment of medicinal plants colonizing abundantly on metal-enriched fly ash deposits: phytoremediation prospective.

Heavy metal-enriched fly ash (FA) deposits are recognized as hazardous contaminated sites on the earth, which pollute our ecosystems. Consequently, the present investigation was carried out to explore the phytoremediation potential of naturally growing medicinal plants in the FA dumpsite. This present study chose two native medicinal plants i.e., Bacopa monnieri and Acmella oleracea found to be naturally colonizing abundantly on FA dumpsite to assess heavy metal accumulation. FA sample of B. monnieri thriving sites found to have metal content in order Mn (216.6)> Cr (39.27)> Zn (20.8)> Ni (16.1)> Cu (15.03)> Co (6.7)> Pb (5.43) whereas for A. oleracea FA dumpsites, the order of metal availability was Mn (750.3)> B (54.5)>Cr (37.2)>Zn (31.33)> Cu (18.7)> Ni (16.93)> Co (7.7)>Pb (4.23). In B. monnieri, higher concentrations of Cr and Mn were observed in the shoot in comparison to the root, indicative of its potential as a hyperaccumulator plant. Conversely, in A. oleracea, greater amounts of Pb were detected in the shoot relative to the root. Hence, it is recommended that B. monnieri and A. oleracea grow on such heavy metal-enriched substrates should be avoided for medicinal purposes; however, these plants can be used for phytoremediation purposes.

Fly ash phytoremediation through natural colonizer plant species is limited.Native colonizing plant species on fly ash has a pivotal role in phytoremediation.Naturally colonizing medicinal plants were dominant over the Fly ash dumpsites.Bacopa monnieri and Acmella oleracea have phytoremediation potential on fly ash.Indeed, fly ash-grown medicinal plants should not be used by local communities.

The Impact of Supratrochlear Foramen on Humeri's Medullary Canal Diameter and Its Surgical Implications.

Introduction Supracondylar elbow fractures are prevalent in the pediatric age group, and retrograde intramedullary nailing of the humerus is a common treatment approach. The anatomy of the medullary canal and the presence of the supratrochlear foramen (STF) significantly influence the stabilization of the nail. This study aimed to determine the incidence and morphology of the STF and compare the width of the medullary canal in humeri with and without the STF. Methods We examined 40 humeri bones with fused humeral epiphyses for the presence of the STF obtained from the Department of Anatomy's osteology collections. We studied the morphology and measured the dimensions of the foramen. We then compared the morphometric characteristics of humeri bearing the STF with those without it. We also took radiographs of all bones and measured the medullary canal width at three levels. We compared the measured width of the medullary canal in humeri between those with an STF and those without. This analysis was performed using IBM SPSS Statistics version 27.0 (Armonk, NY: IBM Corp.) for Windows and an unpaired t-test. We considered p-values of less than 0.05 as statistically significant. Results We found the STF in six bones (15%) as follows: four on the left side and two on the right. Humeri with the STF had smaller mean head circumference, length, and shaft circumference than those without the foramen. However, this difference was not statistically significant (p>0.05). The mean width of the medullary canal in humeri with the STF was significantly smaller (p<0.05) than in bones without the STF. Conclusions Our study revealed that humeri with an STF present a significantly smaller diameter of the medullary canal, which can impact surgical procedures like retrograde intramedullary nailing. Clinicians should consider this when planning surgeries to avoid iatrogenic fractures and enhance procedural efficiency. Our data also suggest that larger humeri are less likely to have an STF, potentially influencing pre-surgical planning and fracture risk assessment.

Generalized rough and fuzzy rough automata for semantic computing.

The classical automata, fuzzy finite automata, and rough finite state automata are some formal models of computing used to perform the task of computation and are considered to be the input device. These computational models are valid only for fixed input alphabets for which they are defined and, therefore, are less user-friendly and have limited applications. The semantic computing techniques provide a way to redefine them to improve their scope and applicability. In this paper, the concept of semantically equivalent concepts and semantically related concepts in information about real-world applications datasets are used to introduce and study two new formal models of computations with semantic computing (SC), namely, a rough finite-state automaton for SC and a fuzzy finite rough automaton for SC as extensions of rough finite-state automaton and fuzzy finite-state automaton, respectively, in two different ways. The traditional rough finite-state automata can not deal with situations when external alphabet or semantically equivalent concepts are given as inputs. The proposed rough finite-state automaton for SC can handle such situations and accept such inputs and is shown to have successful real-world applications. Similarly, a fuzzy finite rough automaton corresponding to a fuzzy automaton is also failed to process input alphabet different from their input alphabet, the proposed fuzzy finite rough automaton for SC corresponding to a given fuzzy finite automaton is capable of processing semantically related input, and external input alphabet information from the dataset obtained by real-world applications and provide better user experience and applicability as compared to classical fuzzy finite rough automaton.

Comparative effectiveness of oral dexamethasone vs. oral prednisolone for acute exacerbation of asthma: A randomized control trial.

Background: Acute exacerbation of asthma is a common condition leading to emergency visits. Prednisolone is a commonly prescribed drug in the standard management of acute exacerbation of asthma along with other drugs. This study was planned to see the efficacy of oral dexamethasone when compared with oral prednisolone in the management of acute exacerbation of asthma. Methods: A single-center pilot study in the form of randomized control trial was done by recruiting children aged 2-14 years diagnosed with acute asthma exacerbation with mild to moderate severity. A total of 88 patients received oral dexamethasone (0.3 mg/kg) in two doses 24 h apart, which was compared with 87 patients who received oral prednisolone (1 mg/kg) in two divided doses 12 h apart for 5 days. The patients were assessed at the time of admission (zero hour), at 4th hour, and on the 5th day by various parameters such as respiratory rate, use of accessory muscles, Pediatric Respiratory Assessment Measure (PRAM) score, peak expiratory flow rate (PEFR), 6-h admission stay, and rate of hospital admission. Results: Baseline demographic profile, clinical characteristics, comorbidities, indoor pollution, and use of Metered Dose Inhaler (MDI) among the two study groups were comparable. Six-hour emergency stay and rate of admission were significantly lower in the dexamethasone group (P < 0.05). Improvement in PRAM score, PEFR, use of accessory muscles, and respiratory rate was also better in dexamethasone group at the 4th hour and 5th day (P < 0.05). In addition, oral dexamethasone was shown to have less incidence of vomiting/gastritis than prednisolone (P < 0.05). Conclusion: Oral dexamethasone can be considered a reliable and better option as compared with prednisolone due to its faster action and minimal side effects.

Study of effect of gluten-free diet on vitamin D levels and bone mineral density in celiac disease patients.

Objective: Celiac disease (CD) is a multifactorial immune-mediated enteropathy caused by a response to ingested gluten. The current available treatment for CD is lifelong gluten-free diet (GFD). This study was done to see the effect of GFD on Vitamin D levels and bone mass density in celiac patients. Methods: A prospective interventional study on newly diagnosed celiac patients was conducted in the Pediatrics department of a tertiary care teaching institute in 2 stages viz. on presentation and after 6 months of GFD. Anthropometric measurements, biochemical investigations, Vitamin D levels, and DEXA scan was done at recruitment and after 6 months of GFD and was analyzed. Results: In newly diagnosed 60 pediatric celiac patients, positive effect of GFD on anthropometry, hemoglobin, Vitamin D levels, DEXA scan parameters was observed. Significant difference was found in Vitamin D levels which increased from baseline 14.85 ± 5.39 to 18.22 ± 5.67 ng/ml after 6 months of GFD (P < 0.05). Significant difference was found in BMD (mean Z-score) which increased from -0.941 ± 0.738 to -0.640 ± 0.60 after 6 months of GFD (P < 0.001). Conclusion: Our study concluded that there is significant increase in vitamin D levels as well as Z-score, bone mass density (BMD) and bone Mass Content (BMC) after 6 months of GFD.

Genital footprints of extragenital tuberculosis in infertile women: Comparison of various diagnostic modalities.

BACKGROUND: Genital tuberculosis (TB) continues to remain an important cause of infertility in women, especially in developing countries. It is mostly consequent to a primary infection elsewhere in the body. The diagnosis is challenging, considering its paucibacillary nature. Although there are many studies on association of genital tuberculosis with infertility, there is paucity of literature on impact of extragenital tuberculosis on fertility of women through involvement of female reproductive organs. The various diagnostic modalities available have limitations and quest is ongoing for the best diagnostic test. METHOD: This was a prospective observational study conducted at the infertility clinic of a tertiary care health facility where 60 infertile women with either tubal factor or unexplained infertility with or without past history of extragenital tuberculosis were enrolled as study subjects or controls respectively. Mantoux test was performed in all women and diagnostic laparo-hysteroscopy was performed in all women to look for any evidence of uterine and/or tubal damage. The peritoneal fluid was sent for GeneXpert and Liquid culture for mycobacterium tuberculosis. Results of Mantoux test, GeneXpert and liquid culture were compared with the laparohysteroscopic findings. RESULT: Of the thirty infertile women in the study group, 27/30 (90%) had a history of pulmonary tuberculosis and 3/30 (10%) had history of tubercular cervical lymphadenopathy. It was observed that Mantoux test was positive (induration >10 mm) in 27/30 (90%) of women in the study group as compared to only 4/30 (13.3%) controls. Abnormal hysteroscopic findings were documented in 26.6% (8/30) study group women as compared to 6.6% (2/30) women in the control group. Similarly, 60% (18/30) of women in the study group had abnormal laparoscopic findings compared to 33% (10/30) in the control group. Seven out of thirty (23.3%) women were positive for GeneXpert in the study group compared to only 1/30 (3.3%) in the control group. Similarly, liquid culture was positive in 6/30 (20%) of women in the study group as compared to 1/30 (3.3%) in the control group. All the above differences were statistically significant. We observed that the sensitivity of Mantoux test (75.8%) stand alone was higher than the other tests combined (50%). However, specificity and positive predictive value (PPV) increases markedly (up to 100%) to when all the three tests are combined. CONCLUSION: The authors conclude that all women presenting with infertility should be screened for a past history of tuberculosis and actively worked up for genital tuberculosis in case the history is positive. The various available tests (Mantoux test, GeneXpert and liquid culture) have their limitations for the diagnosis of genital tuberculosis. Thus an approach of early resort to laparohysteroscopy in suspected patients is desirable so that definitive management may be instituted timely and promptly.

Effect of Maternal Supplementation With Two Different Doses of Vitamin D During Lactation on Vitamin D Status, Anthropometry and Bone Mass of Infants: A Randomized Controlled Trial.

BACKGROUND: There is a high prevalence of vitamin D deficiency (VDD) in exclusively breast-fed infants in the absence of appropriate vitamin D supplementation. OBJECTIVE: To evaluate the efficacy of two doses of maternal vitamin D supplementation on vitamin D levels of mother-infant pairs and to assess its effect on growth parameters (weight, length and head circumference) and bone mass of infants. STUDY DESIGN: Randomized controlled trial. PARTICIPANTS: Lactating mother-infant pairs (n=220). INTERVENTION: Maternal oral vitamin D supplementation in two doses (group 1: 1,20,000 IU/month and group 2: 12,000 IU/month) for 12 months. MAIN OUTCOMES: Main outcomes: Maternal and infant serum 25OHD levels, and infants' growth and bone mass. RESULTS: There was high prevalence of VDD at baseline in mothers (94%) as well as infants (98.5%), which was reduced to 43.1% in (mothers) and 46.5% in infants after 12 months. Significantly higher median (IQR) serum 25OHD levels (ng/mL) were observed among mothers in group 1 compared to group 2 [46 (17-159) vs 18 (6-64); P<0.01] and in infants [36.5 (15-160) vs 17 (7-32); P<0.01]. No significant association was observed between growth parameters or bone mass and serum 25OHD levels of mother or infant between the two groups. Four mothers (3.6%) and two infants (1.8%) in group I had serum 25OHD>100 ng/mL, but without hypercalciuria or hypercalcemia. CONCLUSION: Bolus vitamin D supplementation in the dose of 1,20,000 IU/month was more efficacious in improving maternal and infant vitamin D status at 12 months, as compared to 12,000 IU/month.

Biophysical properties of the isolated spike protein binding helix of human ACE2.

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel ß-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

Design, Synthesis, and Structural Analysis of Cladosporin-Based Inhibitors of Malaria Parasites.

Here we have described a systematic structure activity relationship (SAR) of a set of compounds inspired from cladosporin, a tool compound that targets parasite (Plasmodium falciparum) lysyl tRNA synthetase (KRS). Four sets of analogues, synthesized based on point changes in the chemical scaffold of cladosporin and other logical modifications and hybridizations, were assessed using high throughput enzymatic and parasitic assays along with in vitro pharmacokinetics. Co-crystallization of the most potent compound in our series (CL-2) with PfKRS revealed its structural basis of enzymatic binding and potency. Further, we report that CL-2 has performed better than cladosporin in terms of metabolic stability. It thus represents a new lead for further optimization toward the development of antimalarial drugs. Collectively, along with a lead compound, the series offers insights on how even the slightest chemical modification might play an important role in enhancing or decreasing the potency of a chemical scaffold.

Understanding of the C-H stretch region of infra-red spectroscopy: an analysis of the final state wavefunctions.

The nature of the wavefunctions associated with the final states in the CH stretching region of several medium sized molecules is analysed. The number of optically bright transitions is much larger than the number of CH oscillators present in the molecule, and they are spread over a range of about 300 cm-1. Several of them are clustered together within about 5 cm-1 with near equal intensities. The final states of all these transitions are superpositions of multiple zeroth order states. In almost all of such superpositions, no single zeroth order state has more than 50% weight. Several multiquantum states, with three to four quanta of excitation dominate the final states, with the CH chromophore contributing only a small weightage. Thus the band structure of the CH stretch region is due to several optically bright transitions whose final states are superpositions of low frequency multiquantum states with the CH chromophore contributing only a small weight to make them spectroscopically active.

Ecological restoration of coal fly ash-dumped area through bamboo plantation.

The present study entails the phytoremediation potential of different bamboo species on 5-year-old FA-dumped site near Koradi thermal power plant of Nagpur, Maharashtra, India. The selected FA-dumped site was treated with farmyard manure, press mud, and bio fertilizer followed by plantation of six promising species of bamboo namely Bambusa balcooa Roxb., Dendrocalamus stocksii (Munro.) M. Kumar, Remesh and Unnikrishnan, Bambusa bambos (L.) Voss, Bambusa wamin E.G. Camus, Bambusa vulgaris var. striata (Lodd. ex Lindl.) Gamble, and Bambusa vulgaris var. vittata Riviere and Riviere. The experimental results indicated that the organic input in the FA-dumped site nourished the soil by improving its physico-chemical, and biological characteristics. The results revealed the contamination of the site with different trace elements in varied quantity including Cr (89.29 mg kg-1), Zn (84.77 mg kg-1), Ni (28.84 mg kg-1), Cu (22.91 mg kg-1), Li (19.65 mg kg-1), Pb (13.47 mg kg-1), and Cd (2.35 mg kg-1). A drastic reduction in concentration of heavy metals in FA was observed after 1 year of bamboo plantation as compared to the initial condition. The results showed that bamboo species are good excluders of Ba, Co, Cr, Li, Ni, Mn, and Zn, whereas they are good accumulators of Cd, Pb, and Cu. The values of biochemical parameters, such as pH, total chlorophyll, ascorbic acid (AA), and relative water content of all the bamboo leaves ranged from 5.11-5.70, 1.56-6.33 mg g-1, 0.16-0.19 mg g-1, and 60.23-76.68%, respectively. It is thereby concluded that the bamboo plantation with biofertilizers and organic amendments may indicate adaptive response to environmental pollution on FA-dumped site.

A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2 ß2 or (α2 ß2 )2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

Corn husk derived magnetized activated carbon for the removal of phenol and para-nitrophenol from aqueous solution: Interaction mechanism, insights on adsorbent characteristics, and isothermal, kinetic and thermodynamic properties.

In this study, waste corn husk was used for the synthesis of an effective adsorbent (cornhusk activated carbon, CHAC) and by treating at two different temperatures, 250 °C (CHAC-250) and 500 °C (CHAC-500) to check adsorption efficiency. The synthesized adsorbents were characterized with the help of scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy, Particle size analysis and x-ray diffraction (XRD), which revealed the different properties of the two adsorbents. The synthesized adsorbents were applied for the removal of phenol and p-nitrophenol (PNP) from aqueous solution. CHAC-500 was more efficient than the CHAC-250. The maximum adsorptions of phenol and PNP by CHAC-500 were ∼96% and ∼94%, respectively, while the maximum adsorptions of phenol and PNP by CHAC-250 were ∼81% and ∼84%, respectively. The adsorption processes were best fitted with the Langmuir adsorption isotherm and the pseudo-second-order kinetic model. The adsorption of phenol was an exothermic process, while that of PNP was an endothermic process, on both adsorbents.

Stem cell-based therapies for HIV/AIDS.

One of the current focuses in HIV/AIDS research is to develop a novel therapeutic strategy that can provide a life-long remission of HIV/AIDS without daily drug treatment and, ultimately, a cure for HIV/AIDS. Hematopoietic stem cell-based anti-HIV gene therapy aims to reconstitute the patient immune system by transplantation of genetically engineered hematopoietic stem cells with anti-HIV genes. Hematopoietic stem cells can self-renew, proliferate and differentiate into mature immune cells. In theory, anti-HIV gene-modified hematopoietic stem cells can continuously provide HIV-resistant immune cells throughout the life of a patient. Therefore, hematopoietic stem cell-based anti-HIV gene therapy has a great potential to provide a life-long remission of HIV/AIDS by a single treatment. Here, we provide a comprehensive review of the recent progress of developing anti-HIV genes, genetic modification of hematopoietic stem progenitor cells, engraftment and reconstitution of anti-HIV gene-modified immune cells, HIV inhibition in in vitro and in vivo animal models, and in human clinical trials.

Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector.

RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC.

Mycobacterium tuberculosis Inhibits RAB7 Recruitment to Selectively Modulate Autophagy Flux in Macrophages.

Here we report a novel regulatory mechanism for autophagy-mediated degradation of Mycobacterium tuberculosis (Mtb) and specific strategy exploited by the virulent Mtb to evade it. We show while both avirulent (H37Ra) and virulent (H37Rv) mycobacteria could readily localize to autophagosomes, their maturation into autolysosomes (flux) was significantly inhibited by the latter strain. The inhibition of autophagy flux by the virulent strain was highly selective, as it did not perturb the basal autophagy flux in the macrophages. Selective inhibition of flux of Mtb-containing autophagosomes required virulence regulators PhoP and ESAT-6. We show that the maturation of Mtb-containing autophagosomes into autolysosomes required recruitment of the late endosome marker RAB7, forming the intermediate compartment amphisomes. Virulent Mtb selectively evaded their targeting to the amphisomes. Thus we report a crosstalk between autophagy and phagosome maturation pathway and highlight the adaptability of Mtb, manifested by selective regulation of autophagy flux.

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates.

Other labs have previously reported the ability of adeno-associated virus serotype 9 (AAV9) to cross the blood-brain barrier (BBB). In this report, we carefully characterized variables that might affect AAV9's efficiency for central nervous system (CNS) transduction in adult mice, including dose, vehicle composition, mannitol coadministration, and use of single-stranded versus self-complementary AAV. We report that AAV9 is able to transduce approximately twice as many neurons as astrocytes across the entire extent of the adult rodent CNS at doses of 1.25 × 10¹², 1 × 10¹³, and 8 × 10¹³ vg/kg. Vehicle composition or mannitol coadministration had only modest effects on CNS transduction, suggesting AAV9 crosses the BBB by an active transport mechanism. Self-complementary vectors were greater than tenfold more efficient than single-stranded vectors. When this approach was applied to juvenile nonhuman primates (NHPs) at the middle dose (9-9.5 × 10¹² vg/kg) tested in mice, a reduction in peripheral organ and brain transduction was observed compared to mice, along with a clear shift toward mostly glial transduction. Moreover, the presence of low levels of pre-existing neutralizing antibodies (NAbs) mostly occluded CNS and peripheral transduction using this delivery approach. Our results indicate that high peripheral tropism, limited neuronal transduction in NHPs, and pre-existing NAbs represent significant barriers to human translation of intravascular AAV9 delivery.