Identification of Splice Variants and Isoforms in Transcriptomics and Proteomics.

Alternative splicing is pivotal to the regulation of gene expression and protein diversity in eukaryotic cells. The detection of alternative splicing events requires specific omics technologies. Although short-read RNA sequencing has successfully supported a plethora of investigations on alternative splicing, the emerging technologies of long-read RNA sequencing and top-down mass spectrometry open new opportunities to identify alternative splicing and protein isoforms with less ambiguity. Here, we summarize improvements in short-read RNA sequencing for alternative splicing analysis, including percent splicing index estimation and differential analysis. We also review the computational methods used in top-down proteomics analysis regarding proteoform identification, including the construction of databases of protein isoforms and statistical analyses of search results. While many improvements in sequencing and computational methods will result from emerging technologies, there should be future endeavors to increase the effectiveness, integration, and proteome coverage of alternative splicing events.

Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation.

Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs.

Rapid identification and discrimination of methicillin-resistant Staphylococcus aureus strains via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: Methicillin-resistant Staphylococcus aureus (MRSA) is one of major clinical pathogens responsible for both hospital- and community-acquired infections worldwide. A delay in targeted antibiotic treatment contributes to longer hospitalization stay, higher costs, and increasing in-hospital mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been integrated into the routine workflow for microbial identification over the past decade, and it has also shown promising functions in the detection of bacterial resistance. Therefore, we describe a rapid MALDI-TOF MS-based methodology for MRSA screening with machine-learning algorithms. METHODS: A total of 452 clinical S. aureus isolates were included in this study, of which 194 were MRSA and 258 were methicillin-sensitive S. aureus (MSSA). The mass-to-charge ratio (m/z) features from MRSA and MSSA strains were binned and selected through Lasso regression. These features were then used to train a non-linear support vector machine (SVM) with radial basis function (RBF) kernels to evaluate the discrimination performance. The classifiers' accuracy, sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve (AUC) were evaluated and compared with those from the random forest (RF) model. RESULTS: A total of 2601 unique spectral peaks of all isolates were identified and 38 m/z features were selected for the classifying model. The AUCs of the non-linear RBF-SVM model and the RF model were 0.89 and 0.87, respectively, and the accuracy ranged between 0.86 (RBF-SVM) and 0.82 (RF). CONCLUSIONS: Our study demonstrates that MALDI-TOF MS coupled with machine-learning algorithms could be used to develop a rapid and easy-to-use method to discriminate MRSA from MSSA. Considering that this method is easy to implement in routine microbiology laboratories, it suggests a cost-effective and time-efficient alternative to conventional resistance detection in the future to improve clinical treatment.

PET, image-guided HDAC inhibition of pediatric diffuse midline glioma improves survival in murine models.

Efforts at altering the dismal prognosis of pediatric midline gliomas focus on direct delivery strategies like convection-enhanced delivery (CED), where a cannula is implanted into tumor. Successful CED treatments require confirmation of tumor coverage, dosimetry, and longitudinal in vivo pharmacokinetic monitoring. These properties would be best determined clinically with image-guided dosimetry using theranostic agents. In this study, we combine CED with novel, molecular-grade positron emission tomography (PET) imaging and show how PETobinostat, a novel PET-imageable HDAC inhibitor, is effective against DIPG models. PET data reveal that CED has significant mouse-to-mouse variability; imaging is used to modulate CED infusions to maximize tumor saturation. The use of PET-guided CED results in survival prolongation in mouse models; imaging shows the need of CED to achieve high brain concentrations. This work demonstrates how personalized image-guided drug delivery may be useful in potentiating CED-based treatment algorithms and supports a foundation for clinical translation of PETobinostat.

Rhoifolin Alleviates Inflammation of Acute Inflammation Animal Models and LPS-Induced RAW264.7 Cells via IKKß/NF-κB Signaling Pathway.

Rhoifolin (ROF) is a main effective component in Citrus grandis 'Tomentosa'. ROF has a potential anti-inflammatory activity, but its specific effects and mechanisms have not been studied. This study investigated the anti-inflammatory activity of ROF and searched for its possible molecular mechanisms. A mouse model of acute inflammation was induced by lipopolysaccharide, and the effects of ROF on pathological damages of the lung and liver were observed. Carrageenan-induced paw edema rat model was used to evaluate the effect of ROF on the volume of swelling paw. In LPS-induced RAW264.7 macrophages, the expression levels of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α were measured using ELISA. Real-time PCR was used to measure the mRNA levels of iNOS and CCL2. Western blot was used to detect the activation of IκBα and IKKß in NF-κB signaling pathways. The results showed that ROF accelerated the recoveries of liver and lung tissue damages in acute inflammation mice and inhibited carrageenan-induced paw edema in rats; in addition, ROF significantly suppressed the secretion of TNF-α, IL-1ß, and IL-6 in the serum of rats and mouse model. In LPS-induced RAW264.7 cells, 100 µmol/L ROF enhanced cell viability and suppressed the production of TNF-α, IL-6, and IL-1ß significantly. ROF also decreased the mRNA expression of iNOS and CCL2 and inhibited IκBα and IKKß phosphorylation. In summary, ROF had a potential therapeutic value for inflammation. Our research provided experimental basis for the further development of ROF as an anti-inflammatory drug and for clarifying the anti-inflammatory substance basis of Citrus grandis 'Tomentosa'. Graphical Abstract.

Using 10,000 Fragment Ions to Inform Scoring in Native Top-down Proteomics.

Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.

Inhibition of 3-phosphoglycerate dehydrogenase (PHGDH) by indole amides abrogates de novo serine synthesis in cancer cells.

Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD+ pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC50 assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.

Real-Time, in Vivo Correlation of Molecular Structure with Drug Distribution in the Brain Striatum Following Convection Enhanced Delivery.

The blood-brain barrier (BBB) represents a major obstacle in delivering therapeutics to brain lesions. Convection-enhanced delivery (CED), a method that bypasses the BBB through direct, cannula-mediated drug delivery, is one solution to maintaining increased, effective drug concentration at these lesions. CED was recently proven safe in a phase I clinical trial against diffuse intrinsic pontine glioma (DIPG), a childhood cancer. Unfortunately, the exact relationship between drug size, charge, and pharmacokinetic behavior in the brain parenchyma are difficult to observe in vivo. PET imaging of CED-delivered agents allows us to determine these relationships. In this study, we label different modifications of the PDGFRA inhibitor dasatinib with fluorine-18 or via a nanofiber-zirconium-89 system so that the effect of drug structure on post-CED behavior can accurately be tracked in vivo, via PET. Relatively unchanged bioactivity is confirmed in patient- and animal-model-derived cell lines of DIPG. In naïve mice, significant individual variability in CED drug clearance is observed, highlighting a need to accurately understand drug behavior during clinical translation. Generally, the half-life for a drug to clear from a CED site is short for low molecular weight dasatinib analogs that bare different charge; 1-3 (1, 32.2 min (95% CI: 27.7-37.8), 2, 44.8 min (27.3-80.8), and 3, 71.7 min (48.6-127.6) minutes) and is much longer for a dasatinib-nanofiber conjugate, 5, (42.8-57.0 days). Positron emission tomography allows us to accurately measure the effect of drug size and charge in monitoring real-time drug behavior in the brain parenchyma of live specimens.

Rac-Mediated Macropinocytosis of Extracellular Protein Promotes Glucose Independence in Non-Small Cell Lung Cancer.

Cancer cells can adapt to nutrient poor conditions by rewiring their metabolism and using alternate fuel sources. Identifying these adaptive metabolic pathways may provide novel targets for cancer therapy. Here, we identify a subset of non-small cell lung cancer (NSCLC) cell lines that survive in the absence of glucose by internalizing and metabolizing extracellular protein via macropinocytosis. Macropinocytosis is increased in these glucose independent cells, and is regulated by phosphoinositide 3-kinase (PI3K) activation of Rac-Pak signaling. Furthermore, inhibition of Rac-dependent macropinocytosis blocks glucose-independent proliferation. We find that degradation of internalized protein produces amino acids, including alanine, which generates TCA cycle and glycolytic intermediates in the absence of glucose. In this process, the conversion of alanine to pyruvate by alanine transaminase 2 (ALT2) is critical for survival during glucose starvation. Collectively, Rac driven macropinocytosis of extracellular protein is an adaptive metabolic pathway used by a subset of lung cancers to survive states of glucose deprivation, and may serve as a potential drug target for cancer therapy.

Soft Biomaterial-based Nanocrystal in Pharmaceutical.

BACKGROUND AND OBJECTIVE: Bio-soft material, a class of derivatives of the natural or synthetic material, is getting more and more prevalent in biomedical researches and applications due to the advantages such as in-vivo biodegradation, good water solubility and designable targeting ability. With the presence of bio-soft materials, the drug nanocrystal can be easily generated and aggregated in a feasible process. Given the promising application of the bio-soft material in biological and chemical research, it is valuable to discuss the crucial step in designing bio-soft materials and analyze the emerging properties of bio-soft materials. METHODS: A comprehensive literature survey in the field of bio-soft material development and analysis has been conducted. The collected data and figures were meticulously analyzed and interpreted. RESULTS: In this review, the details of bio-soft materials based nanocrystal were demonstrated in three sections with respect to different materials. In each section, the pros and cons for each bio-soft material and its derivatives were elaborately listed and discussed. CONCLUSION: The review enables an insightful discussion about the properties and the applications of existing biosoft material. It may contribute to the further researches about bio-soft material development and analysis.