Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

Downregulation of barley ferulate 5-hydroxylase dramatically alters straw lignin structure without impact on mechanical properties.

Barley is a major cereal crop for temperate climates, and a diploid genetic model for polyploid wheat. Cereal straw biomass is an attractive source of feedstock for green technologies but lignin, a key determinant of feedstock recalcitrance, complicates bio-conversion processes. However, manipulating lignin content to improve the conversion process could negatively affect agronomic traits. An alternative approach is to manipulate lignin composition which influences the physical and chemical properties of straw. This study validates the function of a barley ferulate 5-hydroxylase gene and demonstrates that its downregulation using the RNA-interference approach substantially impacts lignin composition. We identified five barley genes having putative ferulate 5-hydroxylase activity. Downregulation of HvF5H1 substantially reduced the lignin syringyl/guaiacyl (S/G) ratio in straw while the lignin content, straw mechanical properties, plant growth habit, and grain characteristics all remained unaffected. Metabolic profiling revealed significant changes in the abundance of 173 features in the HvF5H1-RNAi lines. The drastic changes in the lignin polymer of transgenic lines highlight the plasticity of barley lignification processes and the associated potential for manipulating and improving lignocellulosic biomass as a feedstock for green technologies. On the other hand, our results highlight some differences between the lignin biosynthetic pathway in barley, a temperate climate grass, and the warm climate grass, rice, and underscore potential diversity in the lignin biosynthetic pathways in grasses.

Nanoscopic-Assisted Anterior Cruciate Ligament-Posterior Cruciate Ligament Reconstruction.

Knee arthroscopy has allowed us to continue performing surgeries that are minimally invasive and allow patients to have a quick recovery. Multiligamentous knee reconstruction with regards to the anterior cruciate ligament and posterior cruciate ligament can be done in a minimally invasive matter. Visualization is an issue during this surgery, especially looking in the posterior compartment of the knee. The NanoScope (Arthrex, Naples, FL) continues to provide increased possibilities for orthopaedic surgeons. Our technique provides a less-invasive way to observe the posterior compartment to assist the drilling of the tibial tunnel during the posterior cruciate ligament reconstruction. This technique provides distinct advantages over other treatments.

Assessment of Lateral Rotation Percentage and Rotational Deformity of the Elbow in Type 3 Supracondylar Humerus Fractures: A Biomechanical Study.

BACKGROUND: Supracondylar humerus (SCH) fractures are the most common elbow fractures in children. Historically, displaced (Gartland type 3) SCH fractures have been treated with closed reduction and percutaneous pinning. Fluoroscopic imaging is used intraoperatively in order to assess adequate reduction of the fracture fragments before pinning. On lateral fluoroscopic and radiographic images, a lateral rotation percentage (LRP) can be estimated in order to assess rotational deformity. The purpose of this study was to determine the true rotational deformity of distal humerus fracture fragments in SCH fractures based upon the LRP using a clinically relevant laterally based pinning technique. METHODS: In this study, a sawbones model was used to examine the correlation between calculated LRP and the true degree of rotational deformity with 3 of the most common extension-type SCH fracture types (low transverse, high transverse, and lateral oblique). Because fracture stability was not the focus of this study, a single pin was used to hold the construct and allow for fragment rotation along a fixed axis. In this study, 2 of the authors independently measured rotational deformity and compared this with LRP on fluoroscopic lateral imaging of a sawbones model at 0 to 45 degrees of rotational deformity. RESULTS: The LRP of all 3 patterns demonstrated a near linear increase from 0 to 45 degrees with maximum LRP measured at 45 degrees for each of the 3 patterns. Univariate linear regression demonstrated an increase in LRP for the low transverse pattern of 2.02% for every degree of rotation deformity (R2=0.97), 2.29% for the lateral oblique pattern (R2=0.986), and 1.17% for the high transverse pattern (R2=0.971). Maximum LRP was measured at 45 degrees for all 3 patterns with a mean of 53.5% for the high transverse pattern, 93.5% for the low transverse pattern, and 111.2% for the lateral oblique pattern. A higher LRP was measured with increasing degrees of rotational displacement in the low transverse and lateral oblique patterns for all degrees of rotational deformity compared to the high transverse pattern. CONCLUSION: There is a near linear correlation between the degree of malrotation and the LRP with more superior metaphyseal fracture patterns demonstrating a lower LRP than inferior fracture patterns. CLINICAL RELEVANCE: Using our data one can estimate the degree of malrotation based on the LRP on radiographs in the clinical setting. LEVEL OF EVIDENCE: Not applicable (basic-science article).

Conversion of Hip Arthrodesis Using Robotic Arm Technology.

Recent advancements in computer-assisted surgery have led to a renewed interest in robotic-assisted hip arthroplasty. This technology assists with component position which is especially useful in prior trauma or dysplasia cases. We present a case of a surgical hip fusion conversion to total hip arthroplasty with the use of robotic-assisted technology. Enhanced preoperative planning with the ability to manipulate implant position before execution can be invaluable during complex procedures. Further research is warranted before revision cases using computerized navigation systems becomes more prevalent.

Alkaloid chemodiversity in Mandragora spp. is associated with loss-of-functionality of MoH6H, a hyoscyamine 6ß-hydroxylase gene.

Mandrakes (Mandragora spp., Solanaceae) are known to contain tropane alkaloids and have been used since antiquity in traditional medicine. Tropane alkaloids such as scopolamine and hyoscyamine are used in modern medicine to treat pain, motion sickness, as eye pupil dilators and antidotes against organo-phosphate poisoning. Hyoscyamine is converted to 6ß-hydroxyhyoscyamine (anisodamine) and scopolamine by hyoscyamine 6ß-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. We describe here a marked chemo-diversity in the tropane alkaloid content in Mandragora spp. M. officinarum and M. turcomanica lack anisodamine and scopolamine but display up to 10 fold higher hyoscyamine levels as compared with M. autumnalis. Transcriptomic analyses revealed that H6H is highly conserved among scopolamine-producing Solanaceae. MoH6H present in M. officinarum differs in several amino acid residues including a homozygotic mutation in the substrate binding region of the protein and its prevalence among accessions was confirmed by Cleaved-Amplified-Polymorphic-Sequence analyses. Functional expression revealed that MaH6H, a gene isolated from M. autumnalis encodes an active H6H enzyme while the MoH6H sequence isolated from M. officinarum was functionally inactive. A single G to T mutation in nucleotide 663 of MoH6H is associated with the lack of anisodamine and scopolamine in M. officinalis.

A Modern Apparatus for Performing Flash Chromatography: An Experiment for the Organic Laboratory.

A modern apparatus for performing flash chromatography using commercially available, prepacked silica cartridges has been developed. The key advantage of this system, when compared to traditional flash chromatography, is its use of commercially available silica cartridges, which obviates the need for students to handle silica gel. The apparatus has been tested for its ability to perform separations that are commonly found in organic chemistry teaching laboratories, and a laboratory module that combines the techniques of thin-layer chromatography, gas chromatography, and flash chromatography is described. The performance of this new chromatography apparatus was comparable to a traditional flash chromatography column.