Rapid microwave-assisted CNBr cleavage of bead-bound peptides

Large libraries of peptides, cyclic peptides, and other molecules are standard tools for the discovery of drugs, molecular probes, and affinity reagents. In particular, one-bead-one-compound (OBOC) libraries,(1) prepared by the split-and-mix method,(2) provide access to a broad chemical space with a minimum of reagents. Once such a library has been screened against the target of interest, the chemical identity of the library elements on the hit beads is identified. For peptide libraries and their variants, mass spectrometry (MS) based peptide sequencing provides the most rapid method for such analysis. OBOC libraries are constructed in a number of ways to facilitate MS analysis,(3-5) but one common feature is that the peptide must be cleaved from the bead prior to being introduced into the mass spectrometer. While a number of chemical(6) and photochemical(7) cleavage strategies have been developed, the most common strategy is to incorporate a CNBr-cleavable methionine-linker group at the C-terminus of the peptide.(8) CNBr cleavage has also been widely used in proteomics to cleave proteins.(9) With such chemistry, up to 100 beads from an OBOC peptide library can be sequenced in a 24 h period.(10) A large fraction of that time, however, is devoted to the CNBr cleavage step. Standard literature protocols describe CNBr cleavage as requiring between 12 and 24 h, using 20−30 μL of 0.25 M CNBr in 70% aqueous formic acid at room temperature.(11) Although the CNBr cleavage time may be reduced to 2−4 h at elevated temperatures (47 °C), significant side-products may be generated.(12) All reports that we have found that describe CNBr cleavage chemistry from single beads have used the same conditions as for proteomics, although the two chemical processes are not necessarily equivalent

Accurate MALDI-TOF/TOF Sequencing of One-Bead−One-Compound Peptide Libraries with Application to the Identification of Multiligand Protein Affinity Agents Using in Situ Click Chemistry Screening

Combinatorial one-bead−one-compound (OBOC) peptide libraries are widely used for affinity screening, and the sequencing of peptides from hit beads is a key step in the process. For rapid sequencing, CNBr cleavage of the peptides from the beads, followed by de novo sequencing by MALDI-TOF/TOF, is explored. We report on a semiautomated sequencing algorithm and validate it through comparison against Edman degradation sequencing. The initial 44% sequencing success rate of the standard de novo sequencing software was improved to nearly 100%. The sequencing algorithm incorporates existing knowledge of amino acid chemistry and a new strategy for differentiating isobaric amino acids. We tested the algorithm by using MALDI-TOF/TOF to identify a peptide biligand affinity agent against the protein bovine carbonic anhydrase II, starting from comprehensive one-bead−one-compound peptide libraries comprised of non-natural and artificial amino acid components and using the strategy of in situ click/OBOC library screening

Brunner's Gland Hyperplasia: Treatment of Severe Diffuse Nodular Hyperplasia Mimicking a Malignancy on Pancreatic-Duodenal Area

Brunner's gland hyperplasia is a benign tumor of the duodenum and it is rarely associated with clinical symptoms. We report on a 64-yr-old man with Brunner's gland hyperplasia who had undergone a duodenocephalo-pancreatectomy. The reason is that he presented upper gastrointestinal obstructive symptoms and the esophagogastroduodenoscopic finding revealed the lesion to be an infiltrating type mass on the second portion of the duodenum with luminal narrowing. An abdominal computed tomography showed a 2.5 cm-sized mass in the duodenal second portion with a suspicious pancreatic invasion and 7 mm-sized lymph node around the duodenum. Duodenocephalopancreatectomy was successfully performed. Histological examination revealed a Brunner's gland hyperplasia. The final diagnosis was the coexistence of Brunner's gland hyperplasia and pancreatic heterotopia with a pancreatic head invasion. The literature on Brunner's gland hyperplasia is reviewed

New γ\gamma-ray Transitions Observed in 19^{19}Ne with Implications for the 15^{15}O(α\alpha,γ\gamma)19^{19}Ne Reaction Rate

The $^{15}$O($\alpha$,$\gamma$)$^{19}$Ne reaction is responsible for breakout from the hot CNO cycle in Type I x-ray bursts. Understanding the properties of resonances between $E_x = 4$ and 5 MeV in $^{19}$Ne is crucial in the calculation of this reaction rate. The spins and parities of these states are well known, with the exception of the 4.14- and 4.20-MeV states, which have adopted spin-parities of 9/2$^-$ and 7/2$^-$, respectively. Gamma-ray transitions from these states were studied using triton-$\gamma$-$\gamma$ coincidences from the $^{19}$F($^{3}$He,$t\gamma$)$^{19}$Ne reaction measured with GODDESS (Gammasphere ORRUBA Dual Detectors for Experimental Structure Studies) at Argonne National Laboratory. The observed transitions from the 4.14- and 4.20-MeV states provide strong evidence that the $J^\pi$ values are actually 7/2$^-$ and 9/2$^-$, respectively. These assignments are consistent with the values in the $^{19}$F mirror nucleus and in contrast to previously accepted assignments

Key 19^{19}Ne states identified affecting γ\gamma-ray emission from 18^{18}F in novae

Detection of nuclear-decay $\gamma$ rays provides a sensitive thermometer of nova nucleosynthesis. The most intense $\gamma$-ray flux is thought to be annihilation radiation from the $\beta^+$ decay of $^{18}$F, which is destroyed prior to decay by the $^{18}$F($p$,$\alpha$)$^{15}$O reaction. Estimates of $^{18}$F production had been uncertain, however, because key near-threshold levels in the compound nucleus, $^{19}$Ne, had yet to be identified. This Letter reports the first measurement of the $^{19}$F($^{3}$He,$t\gamma$)$^{19}$Ne reaction, in which the placement of two long-sought 3/2$^+$ levels is suggested via triton-$\gamma$-$\gamma$ coincidences. The precise determination of their resonance energies reduces the upper limit of the rate by a factor of $1.5-17$ at nova temperatures and reduces the average uncertainty on the nova detection probability by a factor of 2.1.Comment: 6 pages, 4 figure

An Earth-mass planet in a time of COVID-19: KMT-2020-BLG-0414Lb

We report the discovery of KMT-2020-BLG-0414Lb, with a planet-to-host mass ratio q2 = 0:9- 1:2 × 10-5 = 3-4 q⊕ at 1σ, which is the lowest mass-ratio microlensing planet to date. Together with two other recent discoveries (4. q=q⊕. 6), it fills out the previous empty sector at the bottom of the triangular (log s; log q) diagram, where s is the planet-host separation in units of the angular Einstein radius θE. Hence, these discoveries call into question the existence, or at least the strength, of the break in the mass-ratio function that was previously suggested to account for the paucity of very low-q planets. Due to the extreme magnification of the event, Amax ∼ 1450 for the underlying single-lens event, its light curve revealed a second companion with q3 ∼ 0:05 and j log s3j ∼ 1, i.e., a factor ∼ 10 closer to or farther from the host in projection. The measurements of the microlens parallax ∼E and the angular Einstein radius ∼E allow estimates of the host, planet and second companion masses, (M1;M2;M3) ∼ (0:3M⊙; 1:0M⊙; 17MJ ), the planet and second companion projected separations, (a⊥;2; a⊥;3) ∼ (1:5; 0:15 or 15) au, and system distance DL ∼ 1 kpc. The lens could account for most or all of the blended light (I ∼ 19:3) and so can be studied immediately with high-resolution photometric and spectroscopic observations that can further clarify the nature of the system. The planet was found as part of a new program of high-cadence follow-up observations of high-magnification events. The detection of this planet, despite the considerable difficulties imposed by COVID-19 (two KMT sites and OGLE were shut down), illustrates the potential utility of this program

Defining the actinic keratosis field: a literature review and discussion

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Deletion of the WD40 Domain of LRRK2 in Zebrafish Causes Parkinsonism-Like Loss of Neurons and Locomotive Defect

LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD