De novo gene synthesis by an antiviral reverse transcriptase.

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

Next generation of multispecific antibody engineering.

Multispecific antibodies recognize two or more epitopes located on the same or distinct targets. This added capability through protein design allows these man-made molecules to address unmet medical needs that are no longer possible with single targeting such as with monoclonal antibodies or cytokines alone. However, the approach to the development of these multispecific molecules has been met with numerous road bumps, which suggests that a new workflow for multispecific molecules is required. The investigation of the molecular basis that mediates the successful assembly of the building blocks into non-native quaternary structures will lead to the writing of a playbook for multispecifics. This is a must do if we are to design workflows that we can control and in turn predict success. Here, we reflect on the current state-of-the-art of therapeutic biologics and look at the building blocks, in terms of proteins, and tools that can be used to build the foundations of such a next-generation workflow.

Emergence of RNA-guided transcription factors via domestication of transposon-encoded TnpB nucleases.

Transposon-encoded tnpB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. This widespread gene family was repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas125,6. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of genes encoded by transposable elements, and reveals that RNA-guided transcription factors emerged long before the development of dCas9-based editors.

Transposon-encoded nucleases use guide RNAs to promote their selfish spread.

Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence.

Novel molecular requirements for CRISPR RNA-guided transposition.

CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.

Transposon-encoded nucleases use guide RNAs to selfishly bias their inheritance.

Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS 200 /IS 605 elements undergo 'peel-and-paste' transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements from Geobacillus stearothermophilus that encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration.

CRISPR-associated transposons (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system. On the donor DNA, large mutagenic libraries identified core binding sites recognized by the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications of CAST systems.

Avoiding N-nitrosodimethylamine formation in metformin pharmaceuticals by limiting dimethylamine and nitrite.

Since late 2019, concerns regarding trace levels of the probable human carcinogen N-dimethylnitrosamine (NDMA) in Metformin-containing pharmaceuticals have been an issue if they exceeded the maximum allowable intake of 96 ng/day for a medicine with long-term intake. Here, we report results from an extensive analysis of NDMA content along the active pharmaceutical ingredient (API) manufacturing process as well as two different drug product manufacturing processes. Our findings confirm that Metformin API is not a significant source of NDMA found in Metformin pharmaceuticals and that NDMA is created at those steps of the drug product manufacturing that introduce heat and nitrite. We demonstrate that reduction of nitrite from excipients is an effective means to reduce NDMA in the drug product. Limiting residual dimethylamine in the API has proven to be another important factor for NDMA control as dimethylamine leads to formation of NDMA in the drug products. Furthermore, analysis of historical batches of drug products has shown that NDMA may increase during storage, but the levels reached were not shelf-life limiting for the products under study.

HSF-1 displays nuclear stress body formation in multiple tissues in Caenorhabditis elegans upon stress and following the transition to adulthood.

The transcription factor heat shock factor-1 (HSF-1) regulates the heat shock response (HSR), a cytoprotective response induced by proteotoxic stresses. Data from model organisms has shown that HSF-1 also has non-stress biological roles, including roles in the regulation of development and longevity. To better study HSF-1 function, we created a C. elegans strain containing HSF-1 tagged with GFP at its endogenous locus utilizing CRISPR/Cas9-guided transgenesis. We show that the HSF-1::GFP CRISPR worm strain behaves similarly to wildtype worms in response to heat and other stresses, and in other physiological processes. HSF-1 was expressed in all tissues assayed. Immediately following the initiation of reproduction, HSF-1 formed nuclear stress bodies, a hallmark of activation, throughout the germline. Upon the transition to adulthood, of HSF-1 nuclear stress bodies appeared in most somatic cells. Genetic loss of the germline suppressed nuclear stress body formation with age, suggesting that the germline influences HSF-1 activity. Interestingly, we found that various neurons did not form nuclear stress bodies after transitioning to adulthood. Therefore, the formation of HSF-1 nuclear stress bodies upon the transition to adulthood does not occur in a synchronous manner in all cell types. In sum, these studies enhance our knowledge of the expression and activity of the aging and proteostasis factor HSF-1 in a tissue-specific manner with age.

Incidence of venous thromboembolism after total hip, total knee and hip fracture surgery at Waitemata District Health Board following a peer-reviewed audit.

AIM: The incidence of venous thromboembolism (VTE) following arthroplasty and hip fracture surgery remains an important metric for quality and financial reasons. An audit at our institution between 2006-2010 showed a higher VTE rate than international data did at the time. This study aims to determine rates of DVT and PE in patients undergoing hip and knee arthroplasty and hip fracture surgery at Waitemata District Health Board (Waitemata DHB) between 1 January 2013 and 31 December 2016. METHODS: This study is a retrospective review of all VTE within three months of elective hip or knee replacement or hip fracture surgery. Data were identified for the period between 2013 and 2016 from Waitemata DHB patient databases, including a dedicated VTE database. RESULTS: The current rates of deep vein thrombosis (DVT) and pulmonary embolism (PE) at our institution following hip or knee arthroplasty or hip fracture surgery are 1.5% and 0.6% respectively, a lower rate than 2.3% and 0.9% respectively in 2006-2010. DVTs were significantly more prevalent after hip fracture surgery than after elective hip or knee arthroplasty, and 71% of DVTs were confined to the distal veins. Of the patients undergoing surgery, 93% received post-operative chemoprophylaxis, mainly aspirin or low molecular-weight heparin (LMWH). CONCLUSION: There has been a significant reduction in VTE rates following elective hip and knee joint replacement and hip fracture surgery between the time periods. This occurred over a period when Waitemata DHB introduced a multi-modal, interdisciplinary team approach to VTE prophylaxis utilising enhanced recovery after surgery (ERAS) pathways. These measures may therefore have contributed to the reduction in VTEs.

Frequency of Occurrence of the Rat Lungworm Parasite in the Invasive Island Apple Snail in South Carolina, USA.

The rat lungworm Angiostrongylus cantonensis is a nematode parasite that can cause potentially fatal eosinophilic meningitis in humans. The life cycle of A. cantonensis involves multiple hosts, with the most common terminal hosts being rodents and intermediate hosts comprising gastropods. One such gastropod is the invasive island apple snail Pomacea maculata, which is native to South America but is currently established in several states in the USA, including South Carolina. It has been identified as an intermediate host for A. cantonensis in several locations in Louisiana. The ability of the island apple snail to serve as an intermediate host for A. cantonensis poses significant potential threats to human health, yet no studies to date have determined the prevalence of this parasite in island apple snails in South Carolina. The objective of this study was to investigate the frequency of occurrence of A. cantonensis in South Carolina island apple snails by using a real-time PCR assay. One-hundred individuals from each of three distinct stormwater retention ponds were tested, and no positive detections were found. Determining the prevalence of A. cantonensis in island apple snails is critical in accurately informing the public as to the risks involved in handling and/or consuming island apple snails.

An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology.

Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins >150kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a 'resolution revolution', owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM.

Mutagenetic and electron microscopy analysis of actin filament severing by Cordon-Bleu, a WH2 domain protein.

Cordon-Bleu (Cobl) is a regulator of actin dynamics in neural development and ciliogenesis. Its function is associated with three adjacent actin binding WASP Homology 2 (WH2) domains. We showed that these WH2 repeats confer multifunctional regulation of actin dynamics, which makes Cobl a « dynamizer ¼ of actin assembly, inducing fast turnover of actin filaments and oscillatory polymerization regime via nucleation, severing, and rapid depolymerization activities. Cobl is the most efficient severer of actin filaments characterized so far. To understand which primary sequence elements determine the filament severing activity of the WH2 repeats, here we combine a mutagenetic/domain swapping approach of the minimal fully active Cobl-KAB construct, which comprises the lysine rich region K preceding the two first WH2 domains A and B. The mutated Cobl constructs display variable loss of the original filament nucleating activities of native Cobl-KAB, without any strict correlation with a loss in actin binding, which emphasizes the functional importance of the electrostatic environment of WH2 domains. Filament severing displayed the greatest stringency and was abolished in all mutated forms of Cobl-KAB. Filament severing and re-annealing by Cobl-KAB, which is key in its rapid remodeling of a population of actin filaments, and most likely responsible for its function in ciliogenesis, was analyzed by electron microscopy in comparison with Spire and ADF.

ATP-driven remodeling of the linker domain in the dynein motor.

Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.

Shape and flexibility in the titin 11-domain super-repeat.

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of approximately 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 mum long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the 'large super-repeat'. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 mum long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerized when bound to the thick filament.

Pleistocene glacial refugia across the Appalachian Mountains and coastal plain in the millipede genus Narceus: evidence from population genetic, phylogeographic, and paleoclimatic data.

BACKGROUND: Species that are widespread throughout historically glaciated and currently non-glaciated areas provide excellent opportunities to investigate the role of Pleistocene climatic change on the distribution of North American biodiversity. Many studies indicate that northern animal populations exhibit low levels of genetic diversity over geographically widespread areas whereas southern populations exhibit relatively high levels. Recently, paleoclimatic data have been combined with niche-based distribution modeling to locate possible refugia during the Last Glacial Maximum. Using phylogeographic, population, and paleoclimatic data, we show that the distribution and mitochondrial data for the millipede genus Narceus are consistent with classical examples of Pleistocene refugia and subsequent post-glacial population expansion seen in other organismal groups. RESULTS: The phylogeographic structure of Narceus reveals a complex evolutionary history with signatures of multiple refugia in southeastern North America followed by two major northern expansions. Evidence for refugial populations were found in the southern Appalachian Mountains and in the coastal plain. The northern expansions appear to have radiated from two separate refugia, one from the Gulf Coastal Plain area and the other from the mid-Atlantic coastal region. Distributional models of Narceus during the Last Glacial Maximum show a dramatic reduction from the current distribution, with suitable ecological zones concentrated along the Gulf and Atlantic coastal plain. We found a strong correlation between these zones of ecological suitability inferred from our paleo-model with levels of genetic diversity derived from phylogenetic and population estimates of genetic structuring. CONCLUSION: The signature of climatic change, during and after the Pleistocene, on the distribution of the millipede genus Narceus is evident in the genetic data presented. Niche-based historical distribution modeling strengthens the conclusions drawn from the genetic data and proves useful in identifying probable refugia. Such interdisciplinary biogeographic studies provide a comprehensive approach to understanding these processes that generate and maintain biodiversity as well as the framework necessary to explore questions regarding evolutionary diversification of taxa.

AAA+ Ring and linker swing mechanism in the dynein motor.

Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by approximately 17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.

Biomechanical comparison of unicortical versus bicortical C1 lateral mass screw fixation.

STUDY DESIGN: Biomechanical study of pullout strength of unicortical versus bicortical C1 lateral mass screws using a cadaveric cervical spine model. OBJECTIVE: To compare pullout strength of unicortical versus bicortical C1 lateral mass screws. SUMMARY OF BACKGROUND DATA: The internal carotid artery and hypoglossal nerve lie over the anterior aspect of the lateral mass of the atlas and are at risk from bicortical C1 lateral mass screws. Unicortical screws would reduce the risk of injury to these neurovascular structures; however, no data are available on the relative strength of unicortical versus bicortical C1 lateral mass screws. METHODS: Fifteen cadaveric cervical spine specimens underwent axial pullout testing of C1 lateral mass screws. A unicortical C1 lateral mass screw was placed on 1 side with a contralateral bicortical screw. RESULTS: The mean pullout strengths of the unicortical screws and bicortical screws were 588 N (range, 212 to 1234 N) and 807 N (range, 163 to 1460 N), respectively (P=0.008). CONCLUSIONS: Bicortical C1 lateral mass screws were significantly stronger than unicortical screws; however, the mean pullout strength of both the unicortical and bicortical C1 screws were greater than previously reported values for subaxial lateral mass screws. On the basis of these data, the clinical necessity for using bicortical screw fixation in all patients must be questioned. If similar strength can be achieved using unicortical C1 lateral mass screw to that currently accepted in the subaxial spine, bicortical screws might not be justified for the C1 lateral mass. However, the ability to extrapolate C1-C2 data to subaxial spine data is uncertain because of the difference in normal physiologic loading at these levels.

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state.

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.