Incomplete Cleavage Research Articles

CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication.

Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.

Holoprozencefalie lobară asociată diabetului zaharat matern – prezentare de caz

Holoprosencephaly is a brain abnormality resulting from an incomplete cleavage of the prosencephalon during early embryogenesis. Uncontrolled maternal diabetes mellitus is one of the strongest human teratogens and the most well-known cause of holoprosencephaly. We report a rare case of holoprosencephaly in a male fetus in a preexisting diabetic pregnancy. The diagnosis of the holoprosencephaly was established by ultrasound scan and MRI data.

Heterologous expression and purification of the phage lysin-like bacteriocin LysL from Lactococcus lactis LAC460.

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34mg of protein to be used in future functional studies.

Optimized acid hydrolysis conditions for better characterization the structure of inulin-type fructan from Polygonatum sibiricum

Polygonatum sibiricum is an edible plant species in China known for its abundant polysaccharides. However, correlations between its analytical methods and fine structure have not been established. This is usually due to incomplete cleavage of the glycosidic linkages and instability of hydrolysis. In this study, a new optimal acid hydrolysis method for monosaccharide composition (2 M H2SO4 for 1 h) and methylation analysis (2 mol TFA hydrolysis at 100 °C for 1 h) was developed for characterization of inulin-type fructans, resulting in significantly improved monosaccharide recovery and providing more reliable methylation data. The effectiveness of this method was demonstrated through its application to the study of polysaccharide from P. sibiricum (IPS-70S). The results showed that IPS-70S with a molecular weight of 3.6 kDa is an inulin-type fructans consisting of fructose and glucose in a molar ratio of 27:1. Methylation and NMR analysis indicated that IPS-70S contains →2)-Fruf-(6 → or →2)-Fruf-(1 → with branching →1,6)-Fruf-(2 → and terminates in Glcp-(1 → or Fruf-(2→. In conclusion, optimal acid hydrolysis applicable to the specific polysaccharides contribute to its structurally characterized. The newly optimized acid hydrolysis method for monosaccharide composition and methylation analysis offers a reliable and effective approach to the structural characterization of inulin-type fructans from P. sibiricum. Providing reliable basis for the overall work of NMR analysis and structural analysis, which have potential significance in the field of polysaccharides structural characterization.

Retinal clinical and morphofunctional changes in high myopia in combination with age-related macular degeneration of different stages

Purpose: an analysis of clinical and morphofunctional changes of the retina in high myopia combined with age-related macular degeneration (AMD) of various stages.Materials and methods. We examined 45 patients (87 eyes, mean age 60 years) with high myopia (ave. spherical equivalent (SE) of refraction -11.0 D [-15.0; -7.125]) and the “dry” form of AMD, categories AREDS 1, 2, 3. Additionally, we formed three control groups of the same age range: group 1 consisted of 30 healthy subjects (58 eyes) with a SE between -0.25 D and +0.5 D and no retinal changes; group 2 included 20 patients (38 eyes) with isolated high myopia (SE +0.5 D and no retinal changes; group 2 included 20 patients (38 eyes) with isolated high myopia (SE ³ 6.5 D) and group 3 included another 20 patients (36 eyes) with the “dry” AMD form (AREDS 2, 3). We evaluated the best corrected visual acuity (BCVA), indicators of retinal photosensitivity (MD, PSD) according to computer perimetry data, and morphological characteristics: central retinal thickness (CRT), the anteroposterior axis of the eyeball, the condition of the macular region of the retina according to optical coherence tomography (OCT) in the β-scan mode and autofluorescence (AF).Results: in case of the combined pathology, we detected a reliable reduction of average BCVA to the level of 0.5 [0.3; 0.7] (p < 0.001) and a reduced factor of retinal photosensitivity as compared to the control: MD to -4.36 dB (р<0.001), PSD to 2.97 dB (р < 0.001). CRT was 235 μm (p = 0.122), which showed no statistically significant differences to the control. Morphological changes of the retina corresponded to high myopia and AMD: a dome-shaped profile, lacquer cracks, paravascular retinal microcysts, paravascular lamellar ruptures; myopic maculopathy in the form of an epiretinal membrane, vitreomacular traction due to incomplete vitreoretinal cleavage, myopic foveoshisis; patchy chorioretinal atrophy, areas of diffuse RPE atrophy, damage of the junction line of the outer and inner segments of photoreceptors. The increase in the number and size of drusen depended on the stage of AMD. Normal autofluorescence of the fundus was absent. Pathological AF included minimal changes patterned as focal hypo- and hyperautofluorescence, reticular pattern, focal pattern, linear pattern, lace-like pattern, areas of geographic atrophy with pronounced hypoautofluorescence.Conclusion. Changes in OCT and visual field parameters (MD, PSD) in comorbid pathology (AMD combined with high myopia) were revealed. The morphofunctional indicators were compared for different AMD stages, and the diagnostic significance of AF was established.

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

BackgroundKomagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly.ResultsWe determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein.ConclusionsOur observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.

A plunging ranula in a child with holoprosencephaly: a case of unique pathophysiology and difficult airway management

A ranula is a pseudocyst that originates from the sublingual gland after trauma. Acute cases of ranulas that progress rapidly and cause respiratory distress are rare. Holoprosencephaly is a complex brain malformation caused by incomplete cleavage of the prosencephalon. Children with holoprosencephaly may experience upper airway obstruction due to the associated dentoalveolar malformations and oromotor dysfunctions. We present the case of an eight-year-old female patient with holoprosencephaly and a plunging ranula that manifested as an acute course due to difficult airway management. She required gastrostomy for oromotor dysfunctions related to feeding and swallowing and difficulty managing oral secretions. The sublingual gland and ranula were removed under general anesthesia. Postoperatively, urgent reintubation and close monitoring in the intensive care unit were required due to upper airway obstruction. We successfully managed the patient with close cooperation of a pediatrician and an anesthetist, and no recurrence was observed at the one-year follow-up. A ranula can be caused by trauma to the floor of the mouth in association with lingually inclined mandibular teeth, a type of dentoalveolar compensation seen in maxillary hypoplasia associated with holoprosencephaly. Careful consideration is needed in such cases since airway management can be difficult due to postoperative swelling and oromotor dysfunctions.

Case Report of a Term Newborn with Prenatally Diagnosed Alobar Holoprosencephaly

Abstract Holoprosencephaly is a congenital structural abnormality of brain resulting from incomplete cleavage of prosencephalon into two cerebral hemispheres. It is classified as a disorder of organogenesis and brain histogenesis and is typically associated with facial anomalies. Holoprosencephaly is relatively rare – the incidence in live births varies between 1:10,000 – 1:15,000 but is described in 1 out of 250 spontaneously aborted embryos. There are four major varieties of holoprosencephaly according to the degree of separation of the brain hemispheres; this division is crucial for the patient’s prognosis. Three classic subtypes are alobar, semilobar, and lobar holoprosencephaly and there is also a fourth subtype called middle interhemispheric variant. We present a case study of a term newborn with prenatally diagnosed holoprosencephaly with severe facial dys-morphism and associated health complications.

Construction of a Universal and Label-Free Chemiluminescent Sensor for Accurate Quantification of Both Bacteria and Human Methyltransferases.

DNA methylation plays important roles in various biological processes, and the alteration of DNA methyltransferase activity can induce the aberrant DNA methylation patterns. Despite the progress in methyltransferase activity assays, few methods enable the detection of both bacteria and human methyltransferases. Herein, we construct a universal and label-free chemiluminescent sensor for accurate quantification of both bacteria methyltransferases (e.g., M. SssI methyltransferase (M.SssI MTase)) and human methyltransferases (e.g., DNA (cytosine-5)-methyltransferase 1, (Dnmt1)) by integrating a dumbbell probe with BssHII endonuclease-mediated rolling circle amplification (RCA). We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition, BssHII endonuclease-cleavage, RCA amplification and signal transduction in one probe for the detection of both M.SssI MTase and Dnmt1. Moreover, the introduction of two BssHII endonuclease recognition sites in a dumbbell probe can greatly reduce the false positivity resulting from the incomplete cleavage of dumbbell probe by BssHII, because once one of two recognition sites is identified by BssHII, the dumbbell probe can be completely digested by Exonuclease III (Exo III) and Exonuclease I (Exo I) to prevent the nonspecific RCA. This chemiluminescent sensor can accurately quantify M.SssI MTase in both 10% serum and various cell lysis buffers, and even sensitively detect Dnmt1 activity in MCF-7 cells. Furthermore, this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.SssI MTase, with promising applications in disease diagnosis and drug discovery.

Targeted Bottom-up Characterization of Recombinant Monoclonal Antibodies by Multidimensional LC/MS.

On-line bottom-up approaches have recently emerged as promising alternatives to standard off-line processes for characterizing post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs). The benefits of on-line processing include reductions in required sample amount and sample handling, as well as reducing the overall turnaround time. However, shortening digestion time for the on-line approach of an intact mAb can cause incomplete peptide cleavages, leading to low sequence coverage and poor repeatability of analyses. For the first time, we describe a novel, automated targeted bottom-up strategy consisting of reducing the complexity of intact mAb by digesting the product into small ∼25 kDa fragments, followed by an on-line peptide mapping analysis of each fragment. For this purpose, a four-dimensional-liquid chromatography/mass spectrometry (4D-LC/MS) method was developed using an immobilized IdeS-high-performance liquid chromatography (HPLC) column as a first dimension (1D) for on-line digestion, followed by a (2D) on-column reversed-phase liquid chromatography (RPLC) for reduction and fragments separation. Then, only one fragment was selected for digestion using a (3D) immobilized trypsin cartridge and, finally, the obtained peptides were analyzed by (4D) RPLC-MS. This strategy considerably improved the on-line digestion efficiency with higher sequence coverages (LC and HC >97%), thus allowing various PTMs including oxidation, deamidation, and isomerization located in the complementarity-determining regions (CDRs), as well as N-glycans present on the Fc/2 fragment, to be monitored with similar sensitivity to those obtained with standard off-line approaches. Additional investigations at a middle-up level were also performed via a three-dimensional-LC/MS (3D-LC/MS) approach within the same system, demonstrating the feasibility to achieve a multilevel comprehensive characterization of mAbs.

Synonymous variants in holoprosencephaly alter codon usage and impact the Sonic Hedgehog protein.

Synonymous single nucleotide variants (sSNVs) have been implicated in various genetic disorders through alterations of pre-mRNA splicing, mRNA structure and miRNA regulation. However, their impact on synonymous codon usage and protein translation remains to be elucidated in clinical context. Here, we explore the functional impact of sSNVs in the Sonic Hedgehog (SHH) gene, identified in patients affected by holoprosencephaly, a congenital brain defect resulting from incomplete forebrain cleavage. We identified eight sSNVs in SHH, selectively enriched in holoprosencephaly patients as compared to healthy individuals, and systematically assessed their effect at both transcriptional and translational levels using a series of in silico and in vitro approaches. Although no evidence of impact of these sSNVs on splicing, mRNA structure or miRNA regulation was found, five sSNVs introduced significant changes in codon usage and were predicted to impact protein translation. Cell assays demonstrated that these five sSNVs are associated with a significantly reduced amount of the resulting protein, ranging from 5% to 23%. Inhibition of the proteasome rescued the protein levels for four out of five sSNVs, confirming their impact on protein stability and folding. Remarkably, we found a significant correlation between experimental values of protein reduction and computational measures of codon usage, indicating the relevance of in silico models in predicting the impact of sSNVs on translation. Considering the critical role of SHH in brain development, our findings highlight the clinical relevance of sSNVs in holoprosencephaly and underline the importance of investigating their impact on translation in human pathologies.

Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli

BackgroundAdvantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes.ResultsWe changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173–2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined.ConclusionsOur findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

Development of an efficient LC-MS peptide mapping method using accelerated sample preparation for monoclonal antibodies

Peptide mapping by liquid chromatography-mass spectrometry (LC-MS) is a common method for characterizing primary sequences of monoclonal antibodies (mAbs) and their post-translational modifications (PTMs). Most methods prepare digests by incubating samples with proteases from several hours to overnight. This often induces artifacts of some modifications such as deamidation and isomerization, resulting in overestimated product-related modifications levels. Hour-long-digestion can generate complicate chromatographic profiles due to semi-cleavages or other unnecessary reactions, interfering with the quantitation of peaks of interest. On the other hand, shortening digestion-time can cause incomplete peptide cleavages, thus low sequence coverage and poor repeatability. This study applied pressure cycling technology (PCT) to tryptic digestion and PNGase F deglycosyaltion. A 0.5-h PCT assistant tryptic digestion, by alternating cycles of 10-s atmospheric pressure and 50-s high pressure (30 kpsi) at 37 °C, was evaluated and compared with two conventional digestions, 4-h and 18-h (i.e., overnight) incubations at 37 °C under atmospheric pressure. The 0.5-h PCT assistant deglycosylation was also assessed using the same conditions as those by PCT tryptic digestion. The results demonstrated the application of a 0.5-h PCT to tryptic digestion minimized modification artifacts and reduced interference with quantitation by providing a clean chromatographic profile. The 0.5-h PCT assistant deglycosylation completely removed the N-glycans from the Asn301 in the heavy chains of monoclonal antibody with no impact on the chromatographic profile of the tryptic digests.

Fragments of the Galanin Peptide and Their Synthetic Analogues with the Cardioprotective Effect

New peptide analogs of galanin corresponding to fragments of the N-terminal sequence were synthesized by an automatic solid-phase method using Fmoc-technology. Incomplete cleavage of the Boc-protection from the Trp indole ring was found during postsynthetic procedures and a method for solving this problem was proposed. Physicochemical properties of a number of natural and synthetic N-terminal fragments of galanin were been studied. A comparative evaluation of cardioprotective action of peptide agonists of the galanin GalR2 receptor was carried out on the model of the regional ischemia and reperfusion of the heart in rats in vivo. The analog that contained the carnosine sequence, H-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-βAla-His-OH, was found to most effectively protect the heart from ischemic and reperfusion stress.

Heteropagus Twins with Hepatobiliary and Gastrointestinal Connectedness and Complex Cardiac Malformation in Autosite

Introduction: Heteropagus twins are a set of conjoined twins with one being grossly abnormal, the ‘parasite’, and the other being relatively normal, the ‘autosite’.
 Case Report: A pair of heteropagus twins were initially identified on prenatal ultrasound and subsequently confirmed using prenatal MRI at 25 weeks gestational age. Prenatal imaging identified supernumerary limbs, accessory small bowel loops in the parasitic twin, a hypoplastic left heart in the right hemithorax of the autosite, and a shared liver and bowel between the two. Delivery occurred at 38 + 5 weeks gestation via C-section with an APGAR score of 9/9 for the autosite at both 1 and 5 minutes. MRI confirmed an interconnected autosite-parasite liver that was herniating into the left hemithorax as well as continuous autosite-parasite bowel loops. The patient underwent surgical management including resection of the parasitic twin and repair of the congenital heart defect and is now thriving.
 Discussion: Heteropagus twins include a spectrum of clinical entities from nonconjoined twins to intact conjoined twins. The predominant theory regarding etiology is an incomplete cleavage of a monozygotic embryo at approximately 2 weeks gestation. Following differentiation, ischemia-induced atrophy leads to the embryonic death of the parasite and hemodynamic changes in the autosite to support parasite tissues. The use of antenatal and postnatal imaging is crucial in establishing the diagnosis, management plan, and longterm prognosis secondary to the congenital cardiac malformations and degree of interconnectedness.
 Conclusion: This is the first reported case of heteropagus twins with co-existing hepatobiliary and gastrointestinal continuity.

HIV-1-based Virus-like Particles that Morphologically Resemble Mature, Infectious HIV-1 Virions.

A prophylactic vaccine eliciting both broad neutralizing antibodies (bNAbs) to the HIV-1 envelope glycoprotein (Env) and strong T cell responses would be optimal for preventing HIV-1 transmissions. Replication incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present authentic-structured, virion-associated Env to elicit bNAbs, and also stimulate T cell responses. Here, we optimize our DNA vaccine plasmids as VLP expression vectors for efficient Env incorporation and budding. The original vector that was used in human trials inefficiently produced VLPs, but maximized safety by inactivating RNA genome packaging, enzyme functions that are required for integration into the host genome, and deleting accessory proteins Vif, Vpr, and Nef. These original DNA vaccine vectors generated VLPs with incomplete protease-mediated cleavage of Gag and were irregularly sized. Mutations to restore function within the defective genes revealed that several of the reverse transcriptase (RT) deletions mediated this immature phenotype. Here, we made efficient budding, protease-processed, and mature-form VLPs that resembled infectious virions by introducing alternative mutations that completely removed the RT domain, but preserved most other safety mutations. These VLPs, either expressed from DNA vectors in vivo or purified after expression in vitro, are potentially useful immunogens that can be used to elicit antibody responses that target Env on fully infectious HIV-1 virions.

Cyclopia: A Rare Congenital Malformation

Cyclopia is a rare and lethal congenital anomaly of the forebrain system, resulting from incomplete cleavage of prosencephalon into right and left hemispheres occurring between the 18th and the 28th day of gestation. Approximately 1.05 in 100,000 births are identified as infants with cyclopia, including stillbirths. Many teratogenic factors are identified as the causative factors for this anomaly which include irregular cholesterol biosynthesis, radiation exposure, viruses, alcohol intake and maternal diabetes. Many authors also suggest genetic etiology of this illness. We report a case of 35 year old lady G7P6L5 with previous history of normal vaginal delivery who presented to us in second stage of labor. She delivered a male baby with a large head, a median single eye and absent nose with intact mouth. The baby died soon after the birth. This case is presented because of its rarity. Early ultrasound diagnostics and proper management of this anomaly must be emphasized most strongly to prevent complication associated with this condition.

A practical approach to unveiling auto-catalytic cleavages mediated by Mxe GyrA intein and improving the production of authentic bFGF

A controllable cleavage activity of inteins to release target proteins from affinity tags is an attractive approach for the production of heterologous proteins. Expression of CellBD-GyrA-bfgf (CGF), a fusion precursor comprising the cellulose binding domain (CellBD) of an endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi at the N-terminus, Mxe GyrA intein (GyrA) in the middle, and human basic fibroblast growth factor (bfgf) at the C-terminus, resulted in incomplete auto-catalytic cleavages and a partial release of free bFGF in the cytosol of Escherichia coli. Amino acid substitutions involving Cys1Ala (C1A) and His197Gln (H197Q) performed simultaneously at both termini of GyrA on CellBD-GyrA-bfgf, thus forming the 49-kDa full-length CGF (H197Q; C1A) mutant precursor (49-kDa MP), were found to exhibit an inhibitory effect on the cleavage detected between CellBD and GyrA, thereby enhancing the reservation of intact 49- kDa MP, which was shown to bind well to Avicel. Sequencing analysis showed that 49-kDa MP underwent complex auto-processing activities involving self-cleavages, proteolysis and fusion of degraded fragments to give rise to a variety of smaller intermediates. One such intermediate, 39-kDa (SI), was also found to be able to bind to Avicel. Free bFGF was effectively cleaved from 49-kDa MP and 39-kDa SI bound on Avicel at pH 8.5 or in the presence of succinic acid. Released bFGF was substantiated to possess the authentic structure and potent bioactivity. The findings may pave way for the development of a facile scalable approach for enhancing production of a heterologous protein. Keywords: Mxe GyrA Intein; Site-Specific Mutagenesis; Cellulose Binding Domain; Avicel; bFGF ; in vitro Cleavages; pH 8.5 ; Succinic Acid

Instantaneous Iodine-Assisted DNAzyme Cleavage of Phosphorothioate RNA

Metal ions play a critical role in the RNA-cleavage reaction by interacting with the scissile phosphate and stabilizing the highly negatively charged transition state. Many metal-dependent DNAzymes have been selected for RNA cleavage. Herein, we report that the Ce13d DNAzyme can use nonmetallic iodine (I2) to cleave a phosphorothioate (PS)-modified substrate. The cleavage yield exceeded 60% for both the Rp and Sp stereoisomers in 10 s, while the yield without the enzyme strand was only ∼10%. The Ce13d cleavage with I2 also required Na+, consistent with the property of Ce13d and confirming the similar role of I2 as a metal ion. Ce13d had the highest yield among eight tested DNAzymes, with the second highest DNAzyme showing only 20% cleavage. The incomplete cleavage was due to competition from desulfurization and isomerization reactions. This DNAzyme was engineered for fluorescence-based I2 detection. With EDTA for masking metal ions, I2 was selectively detected down to 4.7 nM. Oxidation of I- with Fe3+ produced I2 in situ, allowing detection of Fe3+ down to 78 nM. By harnessing nonelectrostatic interactions, such as the I2/sulfur interaction observed here, more nonmetal species might be discovered to assist DNAzyme-based RNA cleavage.

Reductive Catalytic Fractionation of C-Lignin

Lignin composed solely of caffeyl alcohol units, or C-lignin, was recently discovered in the seed coats of a number of vanilla orchid and cactus species. The caffeyl alcohol monomer polymerizes into a highly uniform benzodioxane backbone, making C-lignin a promising substrate for lignin valorization, where heterogeneity is a key challenge. In this study, we used reductive catalytic fractionation (RCF) on vanilla seeds to investigate the depolymerization of naturally grown C-lignin. To overcome challenges associated with the high extractive content and poor sugar retention in vanilla seeds, the ratio of monomer yield to total lignin yield was used to isolate the depolymerization efficiency of C-lignin from the extraction efficiency of lignin from seeds. This approach allowed us to compare extents of depolymerization across lignin types and biomass feedstocks. C-Lignin RCF generated extents of depolymerization akin to those of hardwoods, despite observing incomplete benzodioxane cleavage due to catalyst deactivation caused by the seed extractives. In addition, depolymerization of C-lignin produced a favorable monomeric product distribution consisting of only propyl and propenyl catechol. These promising results suggest that genetic modification of other plant species to incorporate C-lignin has the potential to yield a single, valuable catechol product via RCF.