Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1

Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.     

DNA binding and nucleotide flipping by the human DNA repair protein AGT

O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.     

Repair of oligodeoxyribonucleotides by O(6)-alkylguanine-DNA alkyltransferase

Activity of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) is an important source of tumor cell resistance to alkylating agents. AGT inhibitors may prove useful in enhancing chemotherapy. AGT is inactivated by reacting stoichiometrically with O(6)-benzylguanine (b(6)G), which is currently in clinical trials for this purpose. Short oligodeoxyribonucleotides containing a central b(6)G are more potent inactivators of AGT than b(6)G. We examined whether human AGT could react with oligodeoxyribonucleotides containing multiple b(6)G residues. The single-stranded 7-mer 5'-d[T(b(6)G)(5)G]-3' was an excellent AGT substrate with all five b(6)G adducts repaired although one adduct was repaired much more slowly. The highly b(6)G-resistant Y158H and P140K AGT mutants were also inactivated by 5'-d[T(b(6)G)(5)G]-3'. Studies with 7-mers containing a single b(6)G adduct showed that 5'-d[TGGGG(b(6)G)G]-3' was more poorly repaired by wild-type AGT than 5'-d[T(b(6)G)GGGGG]-3' and 5'-d[TGG(b(6)G)GGG]-3' and was even less repairable by mutants Y158H and P140K. This positional effect was unaffected by interchanging the terminal 5'- or 3'-nucleotides and was also observed with single-stranded 16-mer oligodeoxyribonucleotides containing O(6)-methylguanine, where a minimum of four nucleotides 3' to the lesion was required for the most efficient repair. Annealing with the reverse complementary strands to produce double-stranded substrates increased the ability of AGT to repair adducts at all positions except at positions 2 and 15. Our results suggest that AGT recognizes the polarity of single-stranded DNA, with the best substrates having an adduct adjacent to the 5'-terminal residue. These findings will aid in designing novel AGT inhibitors that incorporate O(6)-alkylguanine adducts in oligodeoxyribonucleotide contexts.     

Genotype-dependent differences in S12-RNase expression lead to sporadic self-compatibility

Sporadic self-compatibility, the occasional fruit formation after otherwise incompatible pollinations, has been observed in some S ₁₂-containing genotypes of Solanum chacoense but not in others. We have sequenced this S ₁₂ allele and analyzed its expression in four different genotypes. The S₁₂-RNase levels were generally less abundant than those of other S-RNases present in the same plants. In addition, two-fold and five-fold differences in the amount of S₁₂-RNase and S ₁₂ RNA, respectively, were observed among the genotypes analyzed. A comparison with the genetic data showed that genotypes with the highest levels were fully and permanently self-incompatible, whereas those with the lowest levels were those in which sporadic self-compatibility had been observed. The mature protein contains four potential glycosylation sites and genotype-specific differences in the pattern of glycosylation are also observed. Our results suggest the presence of modifier genes which affect, in a genotype-dependent manner, the level of expression and the post-translational modification of the S₁₂-RNase.     

Characteristics of children admitted to hospital with acute SARS-CoV-2 infection in Canada in 2020

Background:Risk factors for severe outcomes of SARS-CoV-2 infection are not well established in children. We sought to describe pediatric hospital admissions associated with SARS-CoV-2 infection in Canada and identify risk factors for more severe disease.Methods:We conducted a national prospective study using the infrastructure of the Canadian Paediatric Surveillance Program (CPSP). Cases involving children who were admitted to hospital with microbiologically confirmed SARS-CoV-2 infection were reported from Apr. 8 to Dec. 31 2020, through weekly online questionnaires distributed to the CPSP network of more than 2800 pediatricians. We categorized hospital admissions as related to COVID-19, incidental, or for social or infection control reasons and determined risk factors for disease severity in hospital.Results:Among 264 hospital admissions involving children with SARS-CoV-2 infection during the 9-month study period, 150 (56.8%) admissions were related to COVID-19 and 100 (37.9%) were incidental infections (admissions for other reasons and found to be positive for SARS-CoV-2 on screening). Infants (37.3%) and adolescents (29.6%) represented most cases. Among hospital admissions related to COVID-19, 52 (34.7%) had critical disease, 42 (28.0%) of whom required any form of respiratory or hemodynamic support, and 59 (39.3%) had at least 1 underlying comorbidity. Children with obesity, chronic neurologic conditions or chronic lung disease other than asthma were more likely to have severe or critical COVID-19.Interpretation:Among children who were admitted to hospital with SARS-CoV-2 infection in Canada during the early COVID-19 pandemic period, incidental SARS-CoV-2 infection was common. In children admitted with acute COVID-19, obesity and neurologic and respiratory comorbidities were associated with more severe disease.

As of Dec. 31, 2020, Canada had 581 427 confirmed cases of SARS-CoV-2 infection.¹ Similar to other countries, most confirmed infections were in adults, in part because of initial testing policies that targeted older and at-risk populations, as well as prolonged societal containment measures to minimize children’s risk of exposure. Although fewer SARS-CoV-2 infections in children were reported relative to adults during Canada’s first waves of the pandemic,² recent surges in pediatric cases across North America have challenged the notion that children are infected at a lower frequency than adults.^3,4 However, the severity of infection in children appears to be substantially lower, with fewer overall hospital admissions reported and substantially lower mortality compared with adults.^5,6Although risk factors for more severe outcomes of COVID-19 have been well described in adults,⁷ similar risks are less well described in children.⁸ Experience with other viral respiratory infections, including influenza and respiratory syncytial virus, has shown that patient-level factors can increase risk for severe disease in children.^9,10 Understanding populations at risk for severe disease is essential for developing evidence-informed testing strategies, recommendations around reducing exposure (including guidance informing in-person schooling) and potential prioritization of SARS-CoV-2 vaccines in children.To date, few published data have characterized admissions to hospital with SARS-CoV-2 infection among children in Canada. We sought to describe pediatric hospital admissions associated with acute SARS-CoV-2 infection in Canada and identify risk factors for severe disease among children admitted to hospital.     

Giardia lamblia encodes a functional flavohemoglobin

Giardia lamblia is a pathogenic protist that infects the small intestine of mammals. As a facultative anaerobe, Giardia obtains all of its energy by substrate-level phosphorylation, lacks a functioning respiratory chain, and is not thought to require heme. However, sequencing of the G. lamblia genome has identified several putative heme proteins, one of which shares high sequence similarity to flavohemoglobins found in bacteria and some single-celled eukaryotes. We have cloned and characterized the functional properties of the G. lamblia flavohemoglobin. The protein is monomeric, binds heme and flavin adenine dinucleotide, and exhibits similar behavior to known flavohemoglobins, including NADH and NADPH oxidase activity, which is stimulated by addition of the nitric oxide donor DEA/NO. Based on its structural and functional properties, the likely role of this protein is to protect Giardia against oxygen, nitric oxide, or both. The presence of a Giardia gene encoding a functional heme protein raises questions on how this organism acquires the heme cofactor, which hitherto have been unexplored.     

Collagen fiber alignment and maximum principal strain in the glenohumeral capsule predict location of failure during uniaxial extension

The glenohumeral joint is frequently dislocated resulting in injury to the glenohumeral capsule. Repair techniques that focus on restoring the capsule after dislocation to re-establish its stabilizing function could benefit from predictions of the location of failure in this continuous sheet of tissue with a random collagen fiber alignment in the unloaded state. Therefore, the objective of this study was to determine the collagen fiber alignment and maximum principal strain in all regions of the capsule during uniaxial extension to failure and to determine whether these parameters could predict the location of tissue failure. Collagen fiber alignment, quantified using a small-angle light-scattering device, and maximum principal strain in the capsule were determined at 5 % increments of elongation until tissue failure. A contingency table analyzed with Fischer’s exact test demonstrated that peak collagen fiber alignment, represented by the normalized orientation index ( $p < 0.001$ ) and maximum principal strain ( $p < 0.001$ ), is significant in predicting location of failure. The direct correlation between the maximum principal strain and collagen fiber alignment measured prior to failure to the location of tissue failure suggests these parameters can be used as a predictive tool to help locate the areas of the glenohumeral capsule that are susceptible to failure. In the future, changes in collagen fiber alignment following injury could be used to develop a constitutive model for injured capsular tissue.     

Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.     

Chronic Electrical Stimulation with a Suprachoroidal Retinal Prosthesis: A Preclinical Safety and Efficacy Study

Purpose

To assess the safety and efficacy of chronic electrical stimulation of the retina with a suprachoroidal visual prosthesis.

Methods

Seven normally-sighted feline subjects were implanted for 96–143 days with a suprachoroidal electrode array and six were chronically stimulated for 70–105 days at levels that activated the visual cortex. Charge balanced, biphasic, current pulses were delivered to platinum electrodes in a monopolar stimulation mode. Retinal integrity/function and the mechanical stability of the implant were assessed monthly using electroretinography (ERG), optical coherence tomography (OCT) and fundus photography. Electrode impedances were measured weekly and electrically-evoked visual cortex potentials (eEVCPs) were measured monthly to verify that chronic stimuli were suprathreshold. At the end of the chronic stimulation period, thresholds were confirmed with multi-unit recordings from the visual cortex. Randomized, blinded histological assessments were performed by two pathologists to compare the stimulated and non-stimulated retina and adjacent tissue.

Results

All subjects tolerated the surgical and stimulation procedure with no evidence of discomfort or unexpected adverse outcomes. After an initial post-operative settling period, electrode arrays were mechanically stable. Mean electrode impedances were stable between 11–15 kΩ during the implantation period. Visually-evoked ERGs & OCT were normal, and mean eEVCP thresholds did not substantially differ over time. In 81 of 84 electrode-adjacent tissue samples examined, there were no discernible histopathological differences between stimulated and unstimulated tissue. In the remaining three tissue samples there were minor focal fibroblastic and acute inflammatory responses.

Conclusions

Chronic suprathreshold electrical stimulation of the retina using a suprachoroidal electrode array evoked a minimal tissue response and no adverse clinical or histological findings. Moreover, thresholds and electrode impedance remained stable for stimulation durations of up to 15 weeks. This study has demonstrated the safety and efficacy of suprachoroidal stimulation with charge balanced stimulus currents.     

Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C

The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1 mg/mL) increased the motility and viability of sperm preserved for four weeks at 5 °C.