Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition.

Intragenic codon usage in proteobacteria: Translational selection, IS expansion and genomic shrinkage.

This manuscript presents a method to systematically study intragenic variations in codon usage using correspondence analysis and the effective number of codons. The method is applied to >1100 proteobacteria. Codon usage biases (measured as inertia) increases with genome size, the same is true for the percentage of inertia explained by the first axis. It is shown that there is often a relaxed or more uniform codon usage near the gene termini. Ithis is not seen n small genomes, notably those of intracellular organisms like Buchnera aphidicola or Rickettsia prowazekii where translational selection plays less of a role. When genes from E. coli, for which translational selection is well described, are split into low, intermediate and high expression, respectively, it is shown that the intragenic codon usage pattern with more uniform usage at termini exist across all three expression groups. Furthermore, the correspondence analysis reveals a unique pattern in Bordetella pertussis due to IS expansion. This study thus shows that translational selection, genome shrinkage and IS expansion result in characteristic patterns in intragenic codon usage.

Increased Patient's Risk Associated with the Canadian Bioequivalence Guidance Due to Outlier Removal.

BACKGROUND: The Canadian guideline on bioequivalence allows identification of outliers through for example studentized residuals, and it explicitly accepts exclusion of subject data when their studentized residuals reach a certain magnitude. The guideline also requires that the type I errors (patient's risk, chance of declaring bioequivalence for a bioinequivalent product) be maintained at the 5% level. This manuscript investigates if the outlier removal procedure increases type I errors when alpha of 5% is used for construction of the confidence interval. METHODS: Numerical simulation is used. RESULTS: Patient's risk may be inflated beyond 5% when the traditional 90% confidence interval is constructed. Maximum type I errors observed here are slightly above 7%. To circumvent the issue sponsors can either to abstain from outlier removal or find ways to adjust alpha levels downwards if the patient's risk is to be preserved at the 5% level. CONCLUSION: This manuscript is the first to create awareness of a potential inflation of the type I error when 90% confidence intervals are constructed as per Canadian guidance. The issue may not just affect patients in Canada but could also affect patients in the USA since the current rules allow American patients to import a 3 month supply of drugs for personal use and because the central administration is actively pursuing plans to "(…) facilitate the importation of certain prescription drugs that are approved in Canada".

Professor Endrenyi's Legacy: An Evaluation of the Regulatory Requirement "Fixed Effects, Rather Than Random Effects, Should Be Used for All Terms".

PURPOSE: In the latest revision of the guideline for evaluation of bioequivalence (BE), European regulators introduced the requirement for using subjects as fixed factors in the underlying statistical models, even in replicate and semi-replicate studies. The implication was that estimates of within-subject variability were derived with a linear model rather than with a mixed model based on restricted maximum likelihood (REML). While REML-based methods are generally thought to give rise to less biased estimates of variance components, there have been no studies that compared the quality of REML-based estimates and estimates derived via linear models. METHODS: A publication by Endrenyi and Tothfalusi from 1999 described simulations in a fashion that is useful for testing the European Medicines Agency's (EMA) requirement.  This study defines 7 scenarios within which 10,000 individual 2-sequence, 2-treatment, 4-period trials are simulated and makes a comparison of the quality of estimates. RESULTS: It is concluded that estimates based on REML are closer to the true values than estimates based on linear models, but significant differences are only shown in two of the seven scenarios tested.  REML-based estimators have less variability. Both types of estimates appear negatively biased and will therefore decrease the width of the acceptance range.

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

Mitigation of the convergence issues associated with semi-replicated bioequivalence data.

Semi-replicated designs for investigation of bioequivalence constitute a challenge when mixed models are applied. With the commonly available packages and regardless of choice of covariance structure the software may force variance components into the covariance matrix that render it over-specified. This may give rise to arbitrary estimates of certain variance components, lack of convergence or warnings. Classically the covariance matrix is decomposed as V = ZGZt  + R, with G containing the between-subject variance components, Z being the design matrix for the random effects and R containing the within-subject variance components. By abandoning the definitions of G and R, and instead working directly in V, it is possible to specify a correct model with only the variance components of interest. Proof-of-concept for this idea is delivered with a script in the statistical language R. The script is available as supplementary material (Data S1).

Detection of data manipulation in bioequivalence trials.

In recent years regulators have documented how pharmaceutical companies or clinical research organisation can manipulate bioequivalence trial data for non-approvable formulations by performing an interim analysis followed by re-analysis of pharmacokinetic profiles under new subject aliases, with a switch of Test and Reference and/or dilutions. The net effect is that point estimates for failing products will be forced artifically towards 1 and that trials will pass the test for bioequivalence. This is not detectable by any pharmacopoeial method, and is not addressed by common assessment practices at agencies. This paper aims at demonstrating how the signals of such fraudulent study conduct can be detected. The approaches presented are called "Buster" and "SaToWIB" routines; these are computer programs that have been used extensively by regulators to detect signals of fraud but they have not been described in the public domain. The Buster routines visualize trends in the form of partial statistics, residual plots, cumulative confidence intervals, cumulative mean squared errors, and more. Runs tests on the sign of the residuals may constitute a potential test for the manipulation. It is noteworthy that in 2020, regulators in the European Union have publicly begun questioning trial validity on basis of PK profile similarity. The SaToWIB routines rank profile pairs according to numerical similarity on basis of an objective function. It is shown that the rank (as determined by score) is an indicator of fraud in that the actual fraud cases will have higher rank than if there were no relationship between rank and score. The paper also comments on the use of multivariate statistics and discusses the need for development of formal tests for manipulation in view of e.g. multiplicity.

Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein.

A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

Influence of Different Populations on Pharmacokinetic Bioequivalence Results: Can We Extrapolate Bioequivalence Results from One Population to Another?

Purpose: Over the last 15 years, an ever-increasing proportion of pharmacokinetic bioequivalence studies for European/North American generic submissions appeared to have been conducted in geographical/ethnic populations other than those for which the drug is marketed for. The results of pharmacokinetic bioequivalence studies have traditionally been considered to be insensitive to the population studied. However, several recent studies have suggested that this may not necessarily be true. The objective of this study was to investigate whether there were any concerns regarding the current practice of extrapolating bioequivalence study results from one geographic/ethnic population to another. METHODS: In order for a regulatory agency to use bioequivalence results from one population to another, two formulations assessed as bioequivalent under fasted and fed conditions in one population must be bioequivalent in a geographically/ethnically different population under both conditions. Unfortunately, bioequivalence studies between a generic and its reference product for one submission are conducted using only one geographical/ethnic population. As bioequivalence study results between two populations for the same generic and reference products are not available, the food effect for the same reference product between two populations was compared. This is based on the rationale that if two products are bioequivalent under both fasted and fed conditions in two populations, even if there are PK differences in the product exposures between these two populations, the test to reference ratio, as well as the food effect, will remain constant within each population. Food effect (fed/fasted ratio) was calculated using pharmacokinetic data from publicly available regulatory resources and compared between two geographical/ethnic populations using the same reference for each studied drug product. Meta-analyses were conducted. RESULTS: Statistically significant differences (P<0.05) were found in the food effect between two populations for nine out of the ten (90%) available studied products. Among these, an observed clinical difference was suggested in three out of nine (33%) products. CONCLUSION: These results suggest that bioequivalence results from one population may not always be representative of what may be found in another population.

DNA targeting by subtype I-D CRISPR-Cas shows type I and type III features.

Prokaryotic CRISPR-Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR-Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3' and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR-Cas systems.

A Three-Treatment Two-Stage Design for Selection of a Candidate Formulation and Subsequent Demonstration of Bioequivalence.

This paper introduces a two-stage bioequivalence design involving the selection of one out of two candidate formulations at an initial stage and quantifies the overall power (chance of ultimately showing bioequivalence) in a range of scenarios with CVs ranging from 0.1 to 1. The methods introduced are derivates of the methods invented in 2008 by Diane Potvin and co-workers (Pharm Stat. 7(4): 245-262, 2008). The idea is to test the two candidate formulations independently in an initial stage, making a selection of one of these formulations basis of the observed point estimates, and to run, when necessary, a second stage of the trial with pooling of data. Alpha levels are identified which are shown to control the maximum type I error at 5%. Results, expressed as powers and sample sizes, are also published for scenarios where the two formulations are far apart in terms of the match against the reference (one GMR being 0.80, the other GMR being 0.95) and in scenarios where the two test formulations have an actual better match (one GMR being 0.90, the other GMR being 0.95). The methods seem to be compliant with wording of present guidelines from EMA, FDA, WHO, and Health Canada. Therefore the work presented here may be useful for companies developing drugs whose approval hinges on in vivo proof of bioequivalence and where traditional in vitro screening methods (such as dissolution trials) may have poor ability to predict the best candidate.

Structures of the Cmr-ß Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.

Reference Datasets for Studies in a Replicate Design Intended for Average Bioequivalence with Expanding Limits.

In order to help companies qualify and validate the software used to evaluate bioequivalence trials in a replicate design intended for average bioequivalence with expanding limits, this work aims to define datasets with known results. This paper releases 30 reference datasets into the public domain along with proposed consensus results. A proposal is made for results that should be used as validation targets. The datasets were evaluated by seven different software packages according to methods proposed by the European Medicines Agency. For the estimation of CVwR and Method A, all software packages produced results that are in agreement across all datasets. Due to different approximations of the degrees of freedom, slight differences were observed in two software packages for Method B in highly incomplete datasets. All software packages were suitable for the estimation of CVwR and Method A. For Method B, different methods for approximating the denominator degrees of freedom could lead to slight differences, which eventually could lead to contrary decisions in very rare borderline cases.

Pilot and Repeat Trials as Development Tools Associated with Demonstration of Bioequivalence.

The purpose of this work is to use simulated trials to study how pilot trials can be implemented in relation to bioequivalence testing, and how the use of the information obtained at the pilot stage can influence the overall chance of showing bioequivalence (power) or the chance of approving a truly bioinequivalent product (type I error). The work also covers the use of repeat pivotal trials since the difference between a pilot trial followed by a pivotal trial and a pivotal trial followed by a repeat trial is mainly a question of whether a conclusion of bioequivalence can be allowed after the first trial. Repeating a pivotal trial after a failed trial involves dual or serial testing of the bioequivalence null hypothesis, and the paper illustrates how this may inflate the type I error up to almost 10%. Hence, it is questioned if such practice is in the interest of patients. Tables for power, type I error, and sample sizes are provided for a total of six different decision trees which allow the developer to use either the observed geometric mean ratio (GMR) from the first or trial or to assume that the GMR is 0.95. In cases when the true GMR can be controlled so as not to deviate more from unity than 0.95, sequential design methods ad modum Potvin may be superior to pilot trials. The tables provide a quantitative basis for choosing between sequential designs and pivotal trials preceded by pilot trials.

Reference datasets for bioequivalence trials in a two-group parallel design.

In order to help companies qualify and validate the software used to evaluate bioequivalence trials with two parallel treatment groups, this work aims to define datasets with known results. This paper puts a total 11 datasets into the public domain along with proposed consensus obtained via evaluations from six different software packages (R, SAS, WinNonlin, OpenOffice Calc, Kinetica, EquivTest). Insofar as possible, datasets were evaluated with and without the assumption of equal variances for the construction of a 90% confidence interval. Not all software packages provide functionality for the assumption of unequal variances (EquivTest, Kinetica), and not all packages can handle datasets with more than 1000 subjects per group (WinNonlin). Where results could be obtained across all packages, one showed questionable results when datasets contained unequal group sizes (Kinetica). A proposal is made for the results that should be used as validation targets.

Reference datasets for 2-treatment, 2-sequence, 2-period bioequivalence studies.

It is difficult to validate statistical software used to assess bioequivalence since very few datasets with known results are in the public domain, and the few that are published are of moderate size and balanced. The purpose of this paper is therefore to introduce reference datasets of varying complexity in terms of dataset size and characteristics (balance, range, outlier presence, residual error distribution) for 2-treatment, 2-period, 2-sequence bioequivalence studies and to report their point estimates and 90% confidence intervals which companies can use to validate their installations. The results for these datasets were calculated using the commercial packages EquivTest, Kinetica, SAS and WinNonlin, and the non-commercial package R. The results of three of these packages mostly agree, but imbalance between sequences seems to provoke questionable results with one package, which illustrates well the need for proper software validation.