CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable ‘clamped’ Cas12a–DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3′ CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3′ CRISPR RNA to downstream DNA. [Image: see text

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration

Enhancement of transmittance of indium tin oxide coated glass plates.

The transmittance of a glass plate with a transparent conducting coating of a material like indium tin oxide (ITO) can be enhanced by depositing a thin film of a material like silicon dioxide (whose thickness need not be tightly controlled) on top of the transparent conducting coating, avoiding the necessity of designing and depositing a complex multilayer antireflection coating on the opposite surface of the glass plate. The theory behind this performance of the silicon dioxide film and some actual experimental results have been reported

Conformational Dynamics of Single Protein Molecules Studied by Direct Mechanical Manipulation

Advances in single-molecule manipulation techniques have recently enabled researchers to study a growing array of biological processes in unprecedented detail. Individual molecules can now be manipulated with subnanometer precision along a simple and well-defined reaction coordinate, the molecular end-to-end distance, and their conformational changes can be monitored in real time with ever-improving time resolution. The behavior of biomolecules under tension continues to unravel at an accelerated pace and often in combination with computational studies that reveal the atomistic details of the process under investigation. In this chapter, we explain the basic principles of force spectroscopy techniques, with a focus on optical tweezers, and describe some of the theoretical models used to analyze and interpret single-molecule manipulation data. We then highlight some recent and exciting results that have emerged from this research field on protein folding and protein-ligand interactions

Impact of radical nephrectomy on renal functional outcome in patients with no other co-morbidity as determined by 24-h urinary creatinine clearance

Introduction: Renal cell carcinoma (RCC) accounts for 3% of all adult tumors. The mainstay of treatment of RCCs in the past has remained radical nephrectomy (RN). Studies have found a higher cumulative incidence of development of chronic renal insufficiency in patients undergoing RN for RCC. Objectives: To determine the frequency of decrease in 24-h urinary creatinine clearance (CrCl) as an indicator of functional decline after nephrectomy for RCC. Subjects and methods: A total of 103 patients of RCC undergoing RN were included in the study. Patients’ 24-h urinary CrCls were measured pre-nephrectomy and 3 months post-nephrectomy. The patients’ demographic and tumor characteristics were noted from case files. Data was analyzed by using SPSS version 15.0. Results: There were 61 (59.2%) males and 42 (40.8%) females with a mean age of 60.12 ± 8.88 years. The mean maximum tumor diameter was 8.5 ± 2.6 cm. The mean preoperative serum creatinine in the study group was 1.01 ± 0.24 mg/dl, while the mean 3-month postoperative serum creatinine was 1.29 ± 0.46 mg/dl. The mean preoperative CrCl measured in this study was 112.02 ± 6.04 ml/min/1.73 m2, while the 3-month postoperative value was 102.94 ± 14.10 ml/min/1.73 m2, a mean decrease of 9.08 ml/min/1.73 m2. The decrease in CrCl was identified in 34 (33%) patients. No association was found between the measured functional decline and the patients’ age and gender and stage of the disease. Conclusion: The results from this study show that RN is associated with a decrease in CrCl in one third of the study population. Therefore it is recommended that patients undergoing RN should be strictly monitored for occult renal failure and managed promptly to prevent serious morbidity of frank renal failure

5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design

Efficacy and tolerability of tegaserod in constipation dominant irritable bowel syndrome

Objective: To determine the efficacy and tolerability of tegaserod in the treatment of symptoms of irritable bowel syndrome (IBS) IBS-C patients.Design: An open label (quasi interventional) study.PLACE AND DURATION OF STUDY: Patients were enrolled between October 2000 and August 2001 at 4 centres (AKUH, Karachi; Mayo Hospital, Lahore; PIMS, Islamabad; Hayatabad Teaching Complex, Peshawar).PATIENTS AND Methods: Tegaserod was administered in a dose of 6 mg (twice-a-day) orally for a period of 6 weeks. Symptoms were assessed before and during treatment using a questionnaire.Results: The mean age of patients was 37.5 years and 81(69.2%) were males. The study enrolled 117 patients and 101 patients completed the study. Number of bowel movements, symptoms of straining at defecation, stool consistency, bloating, urgency and abdominal pain improved significantly following treatment (p\u3c0.05). Analysis of data in both genders separately showed statistically significant improvement in symptoms of urgency, straining at defecation, abdominal pain and number of bowel movements following treatment. Side effects of diarrhoea and vertigo (6 and 1 patients respectively) necessitating discontinuation of treatment were infrequent.CONCLUSION: Tegaserod given in a dose of 6 mg b.d. is effective and well tolerated in IBS-C patients. It is equally effective in males and females in relieving the symptoms of abdominal pain, bloating, straining at defecation as well as increased in the mean number of bowel movements per week

Protein chain collapse modulation and folding stimulation by GroEL-ES

The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery. BN/Sander Tans La

Structure, Folding Dynamics, and Amyloidogenesis of D76N beta(2)-Microglobulin ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND alpha-CRYSTALLIN

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human beta(2)-microglobulin (beta(2)m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N beta(2)m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type beta(2)m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition

A novel mechano‐enzymatic cleavage mechanism underlies transthyretin amyloidogenesis

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