The origin of mutations is central to understanding evolution and of key relevance to health. Variation occurs non-randomly across the genome, and mechanisms for this remain to be defined. Here, we report that the 5′-ends of Okazaki fragments have significantly elevated levels of nucleotide substitution, indicating a replicative origin for such mutations. With a novel method, emRiboSeq, we map the genome-wide contribution of polymerases, and show that despite Okazaki fragment processing, DNA synthesised by error-prone Pol-α is retained in vivo, comprising ~1.5% of the mature genome. We propose that DNA-binding proteins that rapidly re-associate post-replication act as partial barriers to Pol-δ mediated displacement of Pol-α synthesised DNA, resulting in incorporation of such Pol-α tracts and elevated mutation rates at specific sites. We observe a mutational cost to chromatin and regulatory protein binding, resulting in mutation hotspots at regulatory elements, with signatures of this process detectable in both yeast and humans.

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the master regulator of centriole duplication, the PLK4 kinase, and its substrate TUBGCP6 in patients with microcephalic primordial dwarfism and additional congenital anomalies including retinopathy, extending the human phenotype spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenoptyes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

InterfacesThe quality of a CE separation depends on the CE running buffers, which typically possess high ionic strengths and components of low volatility. However, to obtain a strong and stable ESI signal,

Real‐time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real‐time release (RTR) of biopharmaceuticals. A well‐defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real‐time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated‐data piping, analysis, aggregation, visualization, and smart utility of data for advanced‐data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated‐sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced‐analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

Three different devices that-can be used for surface-induced dissociation (SID) m tandem quadrupole instruments are compared here. The designs were compared by examining the fragmentation of several compounds including benzene, W(CO)6, and (CH3)4N(+). These studies show that SID can be readily implemented on a variety of tandem quadrupoIe instruments and that the spectra obtained with the in-line and 90° instruments are similar. Evidence is presented that confirms that high average internal energies and narrow distributions of internal energy are available by this technique. Efficiencies for fragmentation of odd-electron ions are on the order of those previously reported by others. The overall SID efficiency for even-electron ions is higher than that for odd-electron ions of similar structure.

A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src).

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Specific and nonspecific noncovalent dimer ions of oligonucleotides (ODNs) were observed when mixtures of complementary or noncomplementary strands were analyzed via negative ion electrospray ionization mass spectrometry. Dimer formation was concentration dependent and nearly always occurred when the concentration of ODN exceeded 100 µM. Dimers were observed even for short-length ODNs for which the melting temperature (T m) was well below the experimental temperature and which, therefore, would not be expected to form stable solution duplexes. The abundance of the heterodimer ions seems to correlate with the number of expected hydrogen bonds from Watson-Crick base pairing. As the energy of the incoming ion beam (orifice potential) was increased, the absolute and relative abundance of the dimer ions unexpectedly increased.