The pro-resolving mediator, annexin A1 regulates blood pressure, and age-associated changes in cardiovascular function and remodeling.

Aging is associated with chronic, low-level inflammation which may contribute to cardiovascular pathologies such as hypertension and atherosclerosis. This chronic inflammation may be opposed by endogenous mechanisms to limit inflammation, for example, by the actions of annexin A1 (ANXA1), an endogenous glucocorticoid-regulated protein that has anti-inflammatory and pro-resolving activity. We hypothesized the pro-resolving mediator ANXA1 protects against age-induced changes in blood pressure (BP), cardiovascular structure and function, and cardiac senescence. BP was measured monthly in conscious mature (4-month) and middle-aged (12-month) ANXA1-deficient (ANXA1-/- ) and wild-type C57BL/6 mice. Body composition was measured using EchoMRI, and both cardiac and vascular function using ultrasound imaging. Cardiac hypertrophy, fibrosis and senescence, vascular fibrosis, elastin, and calcification were assessed histologically. Gene expression relevant to structural remodeling, inflammation, and cardiomyocyte senescence were also quantified. In C57BL/6 mice, progression from 4 to 12 months of age did not affect the majority of cardiovascular parameters measured, with the exception of mild cardiac hypertrophy, vascular calcium, and collagen deposition. Interestingly, ANXA1-/- mice exhibited higher BP, regardless of age. Additionally, age progression had a marked impact in ANXA1-/- mice, with markedly augmented vascular remodeling, impaired vascular distensibility, and body composition. Consistent with vascular dysfunction, cardiac dysfunction, and hypertrophy were also evident, together with markers of senescence and inflammation. These findings suggest that endogenous ANXA1 plays a critical role in regulating BP, cardiovascular function, and remodeling and delays cardiac senescence. Our findings support the development of novel ANXA1-based therapies to prevent age-related cardiovascular pathologies.

Opportunities for Fundamental Physics Research with Radioactive Molecules.

Molecules containing short-lived, radioactive nuclei are uniquely positioned to enable a wide range of scientific discoveries in the areas of fundamental symmetries, astrophysics, nuclear structure, and chemistry. Recent advances in the ability to create, cool, and control complex molecules down to the quantum level, along with recent and upcoming advances in radioactive species production at several facilities around the world, create a compelling opportunity to coordinate and combine these efforts to bring precision measurement and control to molecules containing extreme nuclei. In this manuscript, we review the scientific case for studying radioactive molecules, discuss recent atomic, molecular, nuclear, astrophysical, and chemical advances which provide the foundation for their study, describe the facilities where these species are and will be produced, and provide an outlook for the future of this nascent field.

Mixed Epithelial Stromal Tumor of the Kidney With a Mesenteric Lymph Node: A Case Report and Literature Review.

Mixed epithelial and stromal tumor (MEST) of the kidney belongs to the broad spectrum of renal neoplasms, distinguished by their varying composition of stromal to epithelial components. The histopathological display of the biphasic growth pattern of mesenchymal and epithelial elements, often with estrogen and progesterone receptor positivity, clinches the diagnosis. It is typically benign, with low recurrence rates and excellent prognosis after surgical resection. MEST constitutes a rare and unique subset, with limited research and understanding, requiring differentiation from other renal tumors. Our patient's presentation of a morphologically benign renal MEST with an imaging-positive inferior mesenteric lymph node renders this case exceptionally rare.

A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.

CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.

A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination.

CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The data sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.

The Efficacy of the Three Types of Plaque Control Methods During Fixed Orthodontic Treatment: A Randomized Controlled Trial.

BACKGROUND: When intraoral orthodontic devices are used, it becomes significantly more difficult to remove plaque effectively. Dentists and orthodontic specialists can come up with more effective preventive strategies while patients are undergoing fixed orthodontic work if they have a deeper understanding of the present scenario. In addition, individuals will become more aware of the importance of good dental hygiene habits as a result of this. OBJECTIVE: To assess and compare the effectiveness of a manual toothbrush, machine-driven toothbrush, and conventional mechanical toothbrush coupled with mouth rinse in removing plaque and maintaining gingival health in patients undergoing fixed orthodontic treatment. METHODS AND MATERIALS: In this research, a total of 222 individuals who met the eligibility and exclusion requirements were randomly selected and offered their written consent. There were a total of 74 participants for each of the three different categories. Category A used a physically driven toothbrush. Category B used a motorized toothbrush. Category C used a physically driven toothbrush together with mouthwash containing 0.2% chlorhexidine gluconate. All study participants were assessed at baseline, one-month follow-up, and two-month follow-up to document the preliminary information, including that of the modified papillary bleeding index (MPBI) by Muhlemann, plaque index (PI) introduced by Silness and Loe, and gingival index (GI) introduced by Loe and Silness. RESULTS: In this study, the mean PI scores at the one-month and two-month follow-ups were minimum in Category C, while it was maximum in Category A at the two-month follow-up. The mean GI scores at the two-month follow-up were minimum in Category C, while it was maximum in Category A at the two-month follow-up. The mean MPBI scores at the two-month follow-up were minimum in Category C, while it was maximum in Category A. It was observed that participants in this trial who only used a typical mechanical brush experienced an increase in PI and GI scores after one and two months of follow-up. At the one-month and two-month follow-ups, it was noted that the values of PI, GI, and MPBI significantly decreased in the study participants using automated toothbrushes as well as in study participants using manual toothbrushes in conjunction with chlorhexidine mouthwash as compared to baseline values. However, when the three categories were compared, it was found that the research participants utilizing both a manual toothbrush and 0.2% chlorhexidine experienced the highest decreases in PI, GI, and MPBI values. CONCLUSION: The reduction in the scores of PI, GI, and MPBI was maximum in orthodontic patients after two months when they apply manual toothbrushing along with 0.2% chlorhexidine.

Evaluation of the Impact of Chlorhexidine Mouth Rinse on the Bond Strength of Polycarbonate Orthodontic Brackets: A Case-Control Study.

BACKGROUND: Shear bond analysis is the procedure used most frequently to gauge the tensile strength of adhesives incorporated in orthodontic treatments. In shear tensile strength analysis, pressure is placed as close as feasible to the interface between the orthodontic bracket and the surface of the tooth, parallel to the long axis of the tooth. Although numerous research on extracted teeth of human and bovine teeth have been conducted, there may still be variables such as pH, humidity, temperature, and others that could affect how these materials behave in the mouth cavity. The impact of chlorhexidine (CHX) on the binding capacity for non-metallic orthodontic brackets in vivo is not well understood. OBJECTIVE: The goal of the current study is to determine how mouth rinses containing 0.12% CHX affect the adhesive strength of polycarbonate orthodontic brackets. METHODS AND MATERIALS: Thirty-four patients were part of the test category, and they were instructed to wash their oral cavity for approximately 30 seconds using 20 ml of 0.12% CHX gluconate (Septodent). Thirty-four patients made up the control category and were instructed to wash their oral cavity for 30 seconds with a placebo mouthwash of a similar hue (20 ml). Both types of mouthwash were administered to the participants by an administrator who was not specifically involved in the trial and were kept in 120 ml labeled plastic bottles. The study participants were also kept unaware of the type of mouthwash. For the mouthwash utilized by study participants, a double-blinding technique was applied. RESULTS: Thirty-four patients were evaluated in the test category. Since the orthodontic bracket broke in two patients, therefore, 32 patients were evaluated in the control category. The mean value of the strength of the shear bond in the experimental category was 15.32 megapascal (Mpa). The SD value was 2.51. The mean value of the strength of the shear bond in the control subgroup was 15.63. On statistical analysis, the t-value was 0.47. The p-value was 0.671. The difference in findings of the strength of the shear bond was statistically non-significant. CONCLUSION: The results of this investigation allow us to draw the conclusion that the shear bond properties of polycarbonate orthodontic brackets are unaffected when treated with 0.12% CHX preceding the binding. The clinically meaningful adhesion strength was likewise attained by the polycarbonate orthodontic brackets.

Endothelium-dependent relaxation is impaired in Schlager hypertensive (BPH/2J) mice by region-specific mechanisms in conductance and resistance arteries.

AIMS: Endothelial dysfunction and arterial stiffness are hallmarks of hypertension, and major risk factors for cardiovascular disease. BPH/2J (Schlager) mice are a genetic model of spontaneous hypertension, but little is known about the vascular pathophysiology of these mice and the region-specific differences between vascular beds. Therefore, this study compared the vascular function and structure of large conductance (aorta and femoral) and resistance (mesenteric) arteries of BPH/2J mice with their normotensive BPN/2J counterparts. MAIN METHODS: Blood pressure was measured in BPH/2J and BPN/3J mice via pre-implanted radiotelemetry probes. At endpoint, vascular function and passive mechanical wall properties were assessed using wire and pressure myography, qPCR and histology. KEY FINDINGS: Mean arterial blood pressure was elevated in BPH/2J mice compared to BPN/3J controls. Endothelium-dependent relaxation to acetylcholine was attenuated in both the aorta and mesenteric arteries of BPH/2J mice, but through different mechanisms. In the aorta, hypertension reduced the contribution of prostanoids. Conversely, in the mesenteric arteries, hypertension reduced the contribution of both nitric oxide and endothelium-dependent hyperpolarization. Hypertension reduced volume compliance in both femoral and mesenteric arteries, but hypertrophic inward remodelling was only observed in the mesenteric arteries of BPH/2J mice. SIGNIFICANCE: This is the first comprehensive investigation of vascular function and structural remodelling in BPH/2J mice. Overall, hypertensive BPH/2J mice exhibited endothelial dysfunction and adverse vascular remodelling in the macro- and microvasculature, underpinned by distinct region-specific mechanisms. This highlights BPH/2J mice as a highly suitable model for evaluating novel therapeutics to treat hypertension-associated vascular dysfunction.

Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies.

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Förster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with post-transcriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an α-thiophosphorylated variant of rTPT3TP (rTPT3αS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal α-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.

Diagnosis of genitourinary tuberculosis by loop-mediated isothermal amplification based on SYBR Green I dye reaction.

A multitargeted loop-mediated isothermal amplification (MT-LAMP) assay targeting mpt64 (Rv1980c) and IS6110 was designed to diagnose genitourinary tuberculosis (GUTB) cases. While assessing gel-based, hydroxynaphthol blue (HNB) and SYBR Green I MT-LAMP assays on GUTB specimens (n = 28) in a pilot study, both gel-based/SYBR Green I assays exhibited better sensitivity than HNB LAMP. Since SYBR Green MT-LAMP is easier to perform compared with a gel-based assay, a higher number of GUTB specimens (n = 55) were evaluated by SYBR Green MT-LAMP, wherein 85.5% sensitivity and 94.4% specificity (n = 36) were obtained. Moreover, the sensitivity attained by MT-LAMP was significantly higher (p < 0.05) than with multiplex-PCR (mpt64 + IS6110). After further validating these MT-LAMP data in different epidemiological settings, this assay may be developed as a diagnostic kit.

Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM.

CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. A key determinant in Cas9 target selection is DNA duplex unwinding to form an R-loop, in which the single-stranded RNA guide hybridizes with one of the DNA strands. To advance understanding on DNA unwinding by Cas9, we combined two types of spectroscopic label, 2-aminopurine and nitroxide spin-label, to investigate unwinding at a specific DNA base pair induced by Streptococcus pyogenes Cas9. Data obtained with RNA guide lengths varying from 13 to 20 nucleotide revealed that the DNA segment distal to the protospacer adjacent motif can adopt a "partial unwinding" state, in which a mixture of DNA-paired and DNA-unwound populations exist in equilibrium. Significant unwinding can occur at positions not supported by RNA/DNA pairing, and the degree of unwinding depends on RNA guide length and modulates DNA cleavage activity. The results shed light on Cas9 target selection and may inform developments of genome-engineering strategies.

Comparison of Neonatal Outcomes With and Without Prophylaxis With Indomethacin in Premature Neonates.

OBJECTIVE: Historically, prophylactic indomethacin (pINDO) has been used in some institutions for patent ductus arteriosus (PDA) in extremely low birthweight neonates while other institutions have used it as prophylaxis for intraventricular hemorrhage (IVH). The objective of this study was to evaluate the incidence of IVH and PDA with or without pINDO in premature neonates. METHODS: This was a retrospective, single-center study comparing neonatal outcomes in neonates weighing 1250 grams or less who received pINDO (pINDO group) to those who did not (No pINDO group) after our institution discontinued its routine use. RESULTS: A total of 399 infants were included for analysis (pINDO, n = 141; No pINDO, n = 258). No difference was found between pINDO and No pINDO groups in incidence of any IVH (18% vs 14%, respectively) or severe IVH (7% vs 3%, respectively) when adjusting for gestational age and antenatal corticosteroids. Although the incidence of moderate-to-large PDA was lower in the pINDO group (13% vs 23%, respectively, adjusted p = 0.002), there was no significant difference for PDA requiring surgery (4% vs 3%, respectively). Results demonstrated a higher incidence of bronchopulmonary dysplasia (BPD) in the pINDO group (55% vs 41%, respectively, adjusted p = 0.014). CONCLUSION: No difference in the incidence of IVH, severe IVH, or PDA requiring surgery was observed between groups, whereas an increase in BPD was seen with use of pINDO. These data support our institutional practice change to discontinue routine use of pINDO in premature neonates. Further research is needed to guide clinical practice.

Assessment of Oral Health Status of Patients Undergoing Fixed Orthodontics: A Clinical Study.

BACKGROUND: The incidence of dental caries and gingival disease is at higher level in orthodontic patients. The present study demonstrated oral health status of patients undergoing fixed orthodontics. METHODOLOGY: A total of 168 patients age ranged 12-17 years who were undergoing orthodontic treatment for 2 years of both genders were recruited. The assessment of dental caries as Decayed, Missing, and Filled Teeth (DMFT) score and plaque index was determined at first, second, and last visits. RESULTS: Age 12 years had 22 males and 28 females, 13 years had 14 males and 22 females, 14 years had 8 males and 18 females, 15 years had 7 males and 15 females, 16 years had 6 males and 14 females, and 17 years had 9 males and 5 females. The mean DMFT score in age group 12 years was 1.74 and 2.24 at first and third visits, respectively, at 13 years was 1.60 and 2.04 at first and third visits, respectively, at 14 years was 2.38 and 2.72 at first and third visits, respectively, at 15 years was 1.74 and 2.08, at 16 years was 3.32 and 3.56 and at 17 years was 3.40, and 3.64 at first and third visits, respectively. CONCLUSION: There was significant higher dental caries and plaque index in age group 12 years.

Oral Health Related Quality of Life Changes in Standard Cleft and Surgery Patients- A clinical study.

BACKGROUND: Malocclusions are expected to affect subjective attraction, social recognition, and intellect. For dentofacial deformities, functional concerns can also arise. The previous research has established a gradient on oral health-related quality of life (OHRQoL) scores through malocclusion intensity, particularly in the social and emotional realms. This study is used to assess the quality of oral health. MATERIALS AND METHODS: A total of sixty patients began orthodontic therapy at a tertiary-care facility. Treatment in the orthodontic clinic is restricted to serious malocclusions. The study was selected from patients who meet the qualifying requirements of extreme malocclusion and orofacial clefting. The research removed patients with diagnosed hereditary syndromes. Patients got either single-arch or double-arch fixed equipment during their orthodontic procedure. Subjects were categorized as orthodontic patients with extreme malocclusions, needing orthodontic therapy, and severe spinal discrepancies, requiring both orthodontic treatment and orthognatic surgery. The overall score of one subject was 0-56, while the domain score was 0-8. Higher ratings for oral health profiles reflect a stronger effect on the relative quality of life of oral health. RESULTS: For the 14 objects, the mean baseline Oral Health Impact Profile-14 (OHIP-14) score for all three categories was not statistically different for about half of the items. For surgery participants, the OHIP-14 baseline scores were nearly twice as large as the scores of the other two categories for each of these things (P = 0.05). There were a lot of statistically important variations involving the categories, and the three most significant ones are revealing pattern here. The multiple comparison power of nonsignificant predictive variables was extremely weak for the area of physical pain is 5.2%; 41.2% of remaining tests, and 84% for the functional limitation and mental deficiency domain. CONCLUSION: Patients receiving a mixture of orthognathic surgery and orthodontic therapy have comparatively low OHRQoL baseline; however in contrast with normal and cleft patients, they still gain the most from care.

Prolonged Tracheal Intubation and the Association Between Patent Ductus Arteriosus and Bronchopulmonary Dysplasia: A Secondary Analysis of the PDA-TOLERATE trial.

In the PDA-TOLERATE trial, persistent (even for several weeks) moderate to large patent ductus arteriosus (PDA) was not associated with an increased risk of BPD when the infant required <10 days of intubation. However, in infants requiring intubation for ≥10 days, prolonged PDA exposure (≥11 days) was associated with an increased risk of moderate/severe BPD.

NMR spectroscopy analysis reveals differential metabolic responses in arabidopsis roots and leaves treated with a cytokinesis inhibitor.

In plant cytokinesis, de novo formation of a cell plate evolving into the new cell wall partitions the cytoplasm of the dividing cell. In our earlier chemical genomics studies, we identified and characterized the small molecule endosidin-7, that specifically inhibits callose deposition at the cell plate, arresting late-stage cytokinesis in arabidopsis. Endosidin-7 has emerged as a very valuable tool for dissecting this essential plant process. To gain insights regarding its mode of action and the effects of cytokinesis inhibition on the overall plant response, we investigated the effect of endosidin-7 through a nuclear magnetic resonance spectroscopy (NMR) metabolomics approach. In this case study, metabolomics profiles of arabidopsis leaf and root tissues were analyzed at different growth stages and endosidin-7 exposure levels. The results show leaf and root-specific metabolic profile changes and the effects of endosidin-7 treatment on these metabolomes. Statistical analyses indicated that the effect of endosidin-7 treatment was more significant than the developmental impact. The endosidin-7 induced metabolic profiles suggest compensations for cytokinesis inhibition in central metabolism pathways. This study further shows that long-term treatment of endosidin-7 profoundly changes, likely via alteration of hormonal regulation, the primary metabolism of arabidopsis seedlings. Hormonal pathway-changes are likely reflecting the plant's responses, compensating for the arrested cell division, which in turn are leading to global metabolite modulation. The presented NMR spectral data are made available through the Metabolomics Workbench, providing a reference resource for the scientific community.

ZYBT1, a potent, irreversible Bruton's Tyrosine Kinase (BTK) inhibitor that inhibits the C481S BTK with profound efficacy against arthritis and cancer.

Bruton's tyrosine kinase (BTK) plays a central and pivotal role in controlling the pathways involved in the pathobiology of cancer, rheumatoid arthritis (RA), and other autoimmune disorders. ZYBT1 is a potent, irreversible, specific BTK inhibitor that inhibits the ibrutinib-resistant C481S BTK with nanomolar potency. ZYBT1 is found to be a promising molecule to treat both cancer and RA. In the present report we profiled the molecule for in-vitro, in-vivo activity, and pharmacokinetic properties. ZYBT1 inhibits BTK and C481S BTK with an IC50 of 1 nmol/L and 14 nmol/L, respectively, inhibits the growth of various leukemic cell lines with IC50 of 1 nmol/L to 15 µmol/L, blocks the phosphorylation of BTK and PLCγ2, and inhibits secretion of TNF-α, IL-8 and IL-6. It has favorable pharmacokinetic properties suitable for using as an oral anti-cancer and anti-arthritic drug. In accordance with the in-vitro properties, it demonstrated robust efficacy in murine models of collagen-induced arthritis (CIA) and streptococcal cell wall (SCW) induced arthritis. In both models, ZYBT1 alone could suppress the progression of the diseases. It also reduced the growth of TMD8 xenograft tumor. The results suggested that ZYBT1 has high potential for treating RA, and cancer.

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope.

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.

CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity.

Cas12a (also known as "Cpf1") is a class 2 type V-A CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises a single protein and a CRISPR-encoded small RNA (crRNA) and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands. The data revealed that in the absence of complementarity between the crRNA guide and the DNA target-strand, Cas12a binds duplexes with unpaired segments. These off-target duplexes were bound at the Cas12a site responsible for RNA-guided double-stranded DNA binding but were not cleaved due to the lack of RNA/DNA hybrid formation. Such promiscuous binding was attributed to increased DNA flexibility induced by the unpaired segment present next to the protospacer-adjacent-motif. The results suggest that target discrimination of Cas12a can be influenced by flexibility of the DNA. As such, in addition to the linear sequence, flexibility and other physical properties of the DNA should be considered in Cas12a-based genome engineering applications.