Molecular Determinants of Current Blockade Produced by Peptide Transport Through a Nanopore.

The nanopore sensing method holds the promise of delivering a single molecule technology for identification of biological proteins, direct detection of post-translational modifications, and perhaps de novo determination of a protein's amino acid sequence. The key quantity measured in such nanopore sensing experiments is the magnitude of the ionic current passing through a nanopore blocked by a polypeptide chain. Establishing a relationship between the amino acid sequence of a peptide fragment confined within a nanopore and the blockade current flowing through the nanopore remains a major challenge for realizing the nanopore protein sequencing. Using the results of all-atom molecular dynamics simulations, here we compare nanopore sequencing of DNA with nanopore sequencing of proteins. We then delineate the factors affecting the blockade current modulation by the peptide sequence, showing that the current can be determined by (i) the steric footprint of an amino acid, (ii) its interactions with the pore wall, (iii) the local stretching of a polypeptide chain, and (iv) the local enhancement of the ion concentration at the nanopore constriction. We conclude with a brief discussion of the prospects for purely computational prediction of the blockade currents.

Recent development of selective inhibitors targeting the HDAC6 as anti-cancer drugs: Structure, function and design.

HDAC6, a member of the histone deacetylase family, mainly is a cytosolic protein and regulates cell growth by acting on non-histone substrates, such as α -tubulin, cortactin, heat shock protein HSP90, programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), that are closely related to the proliferation, invasion, immune escape and angiogenesis of cancer tissues. The approved drugs targeting the HDACs are all pan-inhibitors and have many side effects due to their lack of selectivity. Therefore, development of selective inhibitors of HDAC6 has attracted much attention in the field of cancer therapy. In this review, we will summarize the relationship between HDAC6 and cancer, and discuss the design strategies of HDAC6 inhibitors for cancer treatment in recent years.

Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing.

DNA is a promising next-generation data storage medium, but challenges remain with synthesis costs and recording latency. Here, we describe a prototype of a DNA data storage system that uses an extended molecular alphabet combining natural and chemically modified nucleotides. Our results show that MspA nanopores can discriminate different combinations and ordered sequences of natural and chemically modified nucleotides in custom-designed oligomers. We further demonstrate single-molecule sequencing of the extended alphabet using a neural network architecture that classifies raw current signals generated by Oxford Nanopore sequencers with an average accuracy exceeding 60% (39× larger than random guessing). Molecular dynamics simulations show that the majority of modified nucleotides lead to only minor perturbations of the DNA double helix. Overall, the extended molecular alphabet may potentially offer a nearly 2-fold increase in storage density and potentially the same order of reduction in the recording latency, thereby enabling new implementations of molecular recorders.

Multiple rereads of single proteins at single-amino acid resolution using nanopores.

A proteomics tool capable of identifying single proteins would be important for cell biology research and applications. Here, we demonstrate a nanopore-based single-molecule peptide reader sensitive to single­amino acid substitutions within individual peptides. A DNA-peptide conjugate was pulled through the biological nanopore MspA by the DNA helicase Hel308. Reading the ion current signal through the nanopore enabled discrimination of single­amino acid substitutions in single reads. Molecular dynamics simulations showed these signals to result from size exclusion and pore binding. We also demonstrate the capability to "rewind" peptide reads, obtaining numerous independent reads of the same molecule, yielding an error rate of <10−6 in single amino acid variant identification. These proof-of-concept experiments constitute a promising basis for the development of a single-molecule protein fingerprinting and analysis technology.

A population-based sex-stratified study to understand how health status preceding traumatic brain injury affects direct medical cost.

OBJECTIVE: To understand how pre-injury health status present five-years preceding traumatic brain injury (TBI) affects direct medical cost two years post-injury. METHODS: Patients age ≥19 years in the emergency department (ED) or acute care for a TBI between April 1, 2007 and March 31, 2014 in Ontario, Canada (N = 55,669) were identified from population-based health administrative data. Forty-three factors of pre-injury health status (i.e., comorbidities and personal, social, and environmental factors) that were internally validated for the TBI population were assessed in this study. The outcome of interest was direct medical cost within two years of discharge. Sex-specific multivariable linear regressions were conducted to understand the associations between direct medical cost within two years of discharge and pre-injury health status. RESULTS: Patients who received care in the ED (81.9% of total sample) incurred a median cost of $2,492/male patient (average $12,342/patient) and $3,508/female patient (average $65,285/patient) within two years of injury; 37 pre-injury factors were significantly associated with increased direct medical costs. Patients who first received care for their TBI in acute care (18.1%) incurred a median cost of $25,081/male patient (average $63,060/patient) and $30,277/female patient (average $65,285/patient) within two years of injury; 21 factors were significantly associated with increased direct medical costs. Among more prevalent factors, those associated with increased medical cost by at least 50% included mental health disorders, substance abuse, disorders or medical conditions frequently observed among the elderly, cardiovascular disorders, stroke and emergencies involving the brain, metabolic disorders and abdominal symptoms, conditions and symptoms of abdomen and pelvis, genitourinary disorders and disorders of prostate, and pulmonary abdominal and other emergencies. CONCLUSIONS: Direct medical costs two years post-TBI differed significantly between patients with and without adverse pre-existing health status. Interdisciplinary teams to promote early identification of pre-existing health conditions and appropriate management and integration of these conditions in TBI care across the continuum of healthcare may be opportunities to reduce direct medical costs post-injury.

Computational Simulation of Adapter Length-Dependent LASSO Probe Capture Efficiency.

Multiplexed cloning of long DNA sequences is a valuable technique in many biotechnology applications, such as long-read genome sequencing and the creation of open reading frame (ORF) libraries. Long-adapter single-stranded oligonucleotide (LASSO) probes have shown promise as a tool to clone long DNA fragments. LASSO probes are molecular inversion probes (MIP) engineered with an adapter region of user-defined length, flanked between template-specific probe sequences. Herein, we demonstrate that the adapter length is a key feature of LASSO that influences the efficiency of gene capture and cloning. Furthermore, we applied a model based on Monte Carlo molecular simulation in order to study the relationship between the long-adapter length of LASSO and capture enrichment. Our results suggest that the adapter length is a factor that contributes to the free energy of target-probe interaction, thereby determining the efficiency of capture. The results indicate that LASSOs with extremely long adapters cannot capture the targets well. They also suggest that targets of different lengths may prefer adapters of different lengths.

Attitudes of Liver Transplant Candidates Toward Organs From Increased-Risk Donors.

Increased-risk donor (IRD) organs make up a significant proportion of the deceased organ donor pool but may be declined by patients on the waiting list for various reasons. We conducted a survey of patients awaiting a liver transplant to determine the factors leading to the acceptance of an IRD organ as well as what strategies could increase the rate of acceptance. Adult liver transplant candidates who were outpatients completed a survey of 51 questions on a 5-point Likert scale with categories related to demographics, knowledge of IRDs, and likelihood of acceptance. A total of 150 transplant candidates completed the survey (age 19-80 years). Male patients constituted 67.3%. Many patients (58.7%) had postsecondary education. Only 23.3% of patients had a potential living donor, and 58/144 (40.3%) were not optimistic about receiving an organ in the next 3 months. The overall IRD organ acceptance rate was 41.1%, whereas 26.2% said they would decline an IRD organ. Women were more likely to accept an IRD organ (54.3% versus 34.7%; P = 0.02). Those who had a college education or higher tended to have lower IRD organ acceptability (28.3% versus 47.4%; P = 0.07). Acceptability also increased as the specified transmission risk of human immunodeficiency virus or hepatitis C virus decreased (P < 0.001). Patients were also more likely to accept an IRD organ if they were educated on the benefits of IRD organs (eg, knowledge that an IRD organ was of better quality increased overall acceptance from 41.1% to 63.3%; P < 0.001). Our survey provides insight into liver transplant candidates who would benefit from greater education on IRD organs. Strategies targeting specific educational points are likely to increase acceptability.

Transcription factors Rv0081 and Rv3334 connect the early and the enduring hypoxic response of Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (M. tb) to survive and persist in the host for decades in an asymptomatic state is an important aspect of tuberculosis pathogenesis. Although adaptation to hypoxia is thought to play a prominent role underlying M. tb persistence, how the bacteria achieve this goal is largely unknown. Rv0081, a member of the DosR regulon, is induced at the early stage of hypoxia while Rv3334 is one of the enduring hypoxic response genes. In this study, we uncovered genetic interactions between these two transcription factors. RNA-seq analysis of ΔRv0081 and ΔRv3334 revealed that the gene expression profiles of these two mutants were highly similar. We also found that under hypoxia, Rv0081 positively regulated the expression of Rv3334 while Rv3334 repressed transcription of Rv0081. In addition, we demonstrated that Rv0081 formed dimer and bound to the promoter region of Rv3334. Taken together, these data suggest that Rv0081 and Rv3334 work in the same regulatory pathway and that Rv3334 functions immediately downstream of Rv0081. We also found that Rv3334 is a bona fide regulator of the enduring hypoxic response genes. Our study has uncovered a regulatory pathway that connects the early and the enduring hypoxic response, revealing a transcriptional cascade that coordinates the temporal response of M. tb to hypoxia.

Discovery of potent, selective, and orally bioavailable inhibitors of interleukin-1 receptor-associate kinase-4.

In this Letter, we report the continued optimization of the N-acyl-2-aminobenzimidazole series, focusing in particular on the N-alkyl substituent and 5-position of the benzimidazole based on the binding mode and the early SAR. These efforts led to the discovery of 16, a highly potent, selective, and orally bioavailable inhibitor of IRAK-4.

N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner.

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies.