Limonoids from the Branches and Leaves of Aglaia lawii and Their Cytotoxic Activities.

Three undescribed limonoids (1-3), named aglaians G-I, and one new natural product azedaralide (4), together with nine known analogues (5-13) were isolated from the branches and leaves of Aglaia lawii by RP C18 column, silica gel column, Sephadex LH-20 column chromatography and preparative HPLC. The structures of the new compounds were elucidated by IR, HRESIMS, 1D, 2D NMR, electronic circular dichroism (ECD) calculations and X-ray crystallography diffraction analysis. The results of bioassay showed that the compound 12 exhibited potential inhibitory activity against six human tumor cell lines (MDA-MB-231, MCF-7, Ln-cap, A549, HeLa and HepG-2) with IC50 values as 8.0-18.6 µM.

Unusual triterpenoids and steroids from Cipadessa baccifera and their biological activities.

Five undescribed triterpenoids and steroids (1-5), as well as ten known compounds, were purified from the branches and leaves of Cipadessa baccifera. Notably, 1 and 2 are rare cipadesin-type limonoids with an unusual 8,30-epoxide ring and 1,8-ether linkage, respectively. Compound 5 possessed pregnane steroid skeleton with an uncommon 5/6/6/6/5-fused ring system. Their structures were constructed by extensive spectroscopic analysis (NMR, IR, UV, and HRESIMS), and their absolute configurations were confirmed by ECD calculations and quantum chemical calculations. All the isolates were in vitro assayed for their antimicrobial potentials against 6 pathogenic microorganisms and antiproliferation activities against five human cancer cell lines. As a result, compounds 5, 12, 13, and 14 exhibited moderate antibacterial activities (MIC: 25-50 µg/mL). Moreover, 5 showed cytotoxicity against five cancer cell lines with IC50 values ranging from 8.0 to 19.9 µM.

Setdb1 -loss induces type-I interferons and immune clearance of melanoma.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1 , as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1 -/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1 -/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1 -/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

Cipacinerasins A-K, structurally diverse limonoids from Cipadessa baccifera.

Eleven undescribed limonoids, cipacinerasins A-K, involving of four diverse carbon skeletal types, along with fifteen known analogues, were isolated from the branches and leaves of Cipadessa baccifera. Within them, cipacinerasins A and B feature a rearranged tetrahydropyranyl ring B formed between C-8 and C-30, are unusual miscellaneous-type limonoids. Cipacinerasins E and F are rare trijugin-type limonoids, of which the D-ring δ-lactone is cleaved. Their structures were elucidated on the basis of extensive spectroscopic data (HRESIMS, NMR, UV and IR), electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analysis. All compounds were evaluated in vitro cytotoxicity against five human tumor cell lines (K562, HeLa, PC3, LN-Cap and Hell), and cipacinerasin E showed moderate antitumor activity with IC50 values ranging from 8.0 to 24.8 µM.

Perinatal outcomes of singleton live births after preimplantation genetic testing during single frozen-thawed blastocyst transfer cycles: a propensity score-matched study.

OBJECTIVE: To determine whether singleton pregnancy achieved after preimplantation genetic testing (PGT) is associated with a higher risk of adverse perinatal outcomes than in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) singleton pregnancy. DESIGN: A retrospective cohort study. SETTING: A university-affiliated fertility center. PATIENT(S): This cohort study included singleton live births resulting from PGT (n = 232) and IVF/ICSI singleton pregnancies (n = 2,829) with single frozen-thawed blastocyst transfer. Multiple baseline covariates were used for propensity score matching, yielding 214 PGT singleton pregnancies matched to 617 IVF/ICSI singleton pregnancies. INTERVENTION(S): Trophectoderm biopsy. MAIN OUTCOME MEASURE(S): The primary outcome was gestational hypertension, and various clinical perinatal secondary outcomes related to maternal and neonatal health were measured. RESULT(S): Compared with IVF/ICSI singleton pregnancy, PGT singleton pregnancy was associated with a significantly higher risk of gestational hypertension (adjusted odds ratio, 2.58; 95% confidence interval, 1.32, 5.05). In the matched sample, the risk of gestational hypertension remained higher with PGT singleton pregnancy (odds ratio, 2.33; 95% confidence interval, 1.04, 5.22) than with IVF/ICSI singleton pregnancy. No statistical differences were noted in any other measured outcomes between the groups. CONCLUSION(S): The perinatal outcomes of PGT and IVF/ICSI singleton pregnancies were similar except for the observed potentially higher risk of gestational hypertension with PGT singleton pregnancy. However, because the data on PGT singleton pregnancies are limited, this conclusion warrants further investigation.

TAGLN mediated stiffness-regulated ovarian cancer progression via RhoA/ROCK pathway.

BACKGROUND: Ovarian cancer (OC) progression is an unmet medical challenge. Since omental metastases were palpated harder than their primary counterparts during cytoreductive surgery of patients with epithelial ovarian cancer (EOC), we were inspired to investigate OC progression from the perspective of biomechanics. METHODS: Atomic Force Microscope (AFM) was used to measure the Young's modulus of tissues. The collagen-coated polyacrylamide hydrogel (PA gel) system was prepared to mimic the soft and stiff substrates in vitro. The effect of TAGLN was evaluated both in vitro and in vivo using transwell assay, immunofluorescence, western blot analysis and immunohistochemistry. RESULTS: We quantitatively confirmed that omental metastases were stiffer and more abundant in desmoplasia compared with paired primary tumors, and further demonstrated that matrix stiffness could notably regulate OC progression. Remarkably, TAGLN, encoding an actin cross-linking/gelling protein, was identified as a potent mechanosensitive gene that could form a regulation loop with Src activation reacting to environmental stiffness, thus mediating stiffness-regulated OC progression through regulating RhoA/ROCK pathway. CONCLUSIONS: These data demonstrate that targeting extra-cellular matrix (ECM) stiffness could probably hamper OC progression, and of note, targeting TAGLN might provide promising clinical therapeutic value for OC therapy.

PPP6C negatively regulates oncogenic ERK signaling through dephosphorylation of MEK.

Flux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.

Analysis of the screening results of 24040 potential sperm donors in a human sperm bank in Henan Province, China: a 14-year retrospective cohort study.

STUDY QUESTION: Is there a relation between the characteristics of potential sperm donors and the acceptance rate of these potential donors? SUMMARY ANSWER: A relatively higher acceptance rate was observed for potential sperm donors who were aged ≤ 35 years, were married, had children, and who had received higher education, and acceptance rates were also higher during spring and winter than summer and autumn. WHAT IS KNOWN ALREADY: Recruiting donors to a sperm bank program is difficult and slow owing to the high rates of rejection and dropout. STUDY DESIGN, SIZE, DURATION: A total of 24040 potential sperm donors were screened by the Henan Human Sperm Bank from 2006 to 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Potential sperm donors were recruited using the following baseline requirement: height of 168 cm or taller; age 22-45 years; currently attending or had graduated from high school or above. Men who met the criteria for age, height, and education level were invited for semen quality screening. The acceptable criteria for semen samples included liquefaction time < 60min, volume ≥ 2mL, sperm concentration ≥ 60 × 106/mL, progressive motility ≥ 60%, post-thaw motility ≥ 40%, pre-freezing total motile sperm per vial > 30 × 106/mL, post-thaw total motile sperm per vial > 12 × 106/mL, and freeze-thaw survival rate ≥ 60%. Any potential sperm donors meeting the minimum criteria for acceptable semen quality on two consecutive semen samples were scheduled for clinical assessment, physical examination, and laboratory tests. The reasons for sperm donor rejection were analyzed. The characteristics of accepted and rejected donors were compared using the chi-square test, and multivariate logistic regression analyses were conducted to identify factors associated with the acceptance rate of potential sperm donors and the positive rate of sexually transmitted diseases (STDs). MAIN RESULTS AND THE ROLE OF CHANCE: Only 23.38% (5620/24040) of potential sperm donors were accepted. The top four reasons for rejection were suboptimal semen quality (90.27%), STDs (6.26%), dropped out (2.65%), and chromosomal abnormalities (0.35%). The most common reason for the rejection of donors with an STD was a positive test for mycoplasmas (49.05%), followed by hepatitis B virus (27.56%), Chlamydia trachomatis (4.68%), and Escherichia coli (3.03%). n this study, the acceptance rate for men aged ≤ 35 years was significantly higher than that for men aged >35 years (P < 0.05). The acceptance rates were also significantly higher for men with a higher education than for men with lower education, married men than unmarried men, and men with children than men without children (P < 0.05). Moreover, acceptance rates were significantly higher during spring and winter than during summer (P <0.05) but were not significantly higher during autumn than during summer (P >0.05). LIMITATIONS, REASONS FOR CAUTION: This study was not performed to analyze the effect of lifestyle habits, such as alcohol consumption and cigarette smoking, on the acceptance rate of potential sperm donors. WIDER IMPLICATIONS OF THE FINDINGS: Only a small proportion of potential sperm donors were accepted in this anonymous sperm donor program. New strategies for sperm donor recruitment may be required to improve the acceptance rate. In the future, we may have to target potential sperm donors who are aged ≤ 35 years and who received higher education in order to improve the acceptance rate. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Joint Construction Project of Henan Medical Science and Technology Research Plan under grant number LHGJ20190389. The authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Anti-tobacco mosaic virus (TMV) activity of chemical constituents from the seeds of Sophora tonkinensis.

Two new compounds, including one new arylbenzofuran (1) and one new pterocarpanoid (2), along with nine known ones, were isolated from the seeds of Sophora tonkinensis. The structures of the new compounds were elucidated based on a comprehensive spectroscopic data analysis. Compounds 2 and 3 exhibited good anti-tobacco mosaic virus (TMV) activities with the protective inhibition rate of 69.62% and 68.72% respectively, at concentration of 100 µg/ml.

Association between morphologic grading and implantation rate of Euploid blastocyst.

BACKGROUND: Standard morphologic evaluation has been the most widely adopted approach to embryo selection, and remains the most common strategy.The objective of the study to determine the association between the morphologic grading and implantation rate of euploid blastocysts in single frozen-thawed embryo transfer (SET) cycles. METHODS: A total of 271 patients aged 20-40 years undergoing euploid SET from January 2017 to December 2019 were included in retrospective cohort study.The cycles were divided into three groups based on their morphologic grading before cryopreservation: good-quality (n = 58), average-quality (n = 88) and poor-quality blastocysts (n = 125). The pregnancy outcome of the three morphologic groups were analyzed and a logistic regression of implantation rate was conducted. RESULTS: Good-quality blastocysts yielded statistically significantly higher implantation rates than poor-quality (79.31% vs. 48%; P<0.001). Planned subgroup analyses by age and the day of TE biopsy were conducted. Logistic regression analyses that adjusted for these variables identified higher implantation rates (adjusted odds ratio(aOR) = 4.083, 95% confidence interval (CI):1.836-9.082, P<0.001) for the good-quality blastocysts than for those that underwent poor-quality cycles in women aged < 35 years, but not in women aged ≥35 years (aOR = 6.074, 95% CI: 0.456-80.919, P = 0.172). The implantation rates were higher among women with good-quality blastocysts on both Day 5 and Day 6 of TE biopsy than among those with poor-quality blastocysts (Day 5, aOR = 3.294, 95% CI:1.260-8.616, P = 0.015; Day 6, aOR = 4.179, 95% CI:1.004 ~ 17.399, P = 0.049). Day 5 euploid blastocysts had no significant difference in implantation potential and early spontaneous abortion rate compared with similarly graded Day 6 euploid blastocysts. CONCLUSIONS: Blastocyst morphologic grading was associated with implantation rate for euploid embryo transfers after adjustment for potential confounders. These findings suggest that evaluating blastocyst morphology is critical when selecting the best euploid blastocyst.

Polycyclic polyprenylated acylphloroglucinol derivatives with neuroprotective effects from Hypericum monogynum.

A new polycyclic polyprenylated acylphloroglucinol (PPAP), hypermonin C (1), along with nine known PPAPs (2-10) were obtained from the leaves and twigs of Hypericum monogynum. The structures of the isolates were determined on the basis of extensive spectroscopic analysis. The neuroprotective effects of the isolates against several chemical-induced injuries in SH-SY5Y and PC12 cells were assessed, and most of the compounds exhibited significant protective effects at 10 µg/ml. Especially, three compounds (1, 3, and 7) showed excellent neuroprotective activity with a cell viability of 92.4% ∼ 95.8% in KCl-induced SH-SY5Y cell injury. Their preliminary structure-activity relationship was also discussed and the configuration of substituent in furohyperforin may be critical for the neuroprotective activity of PPAP derivatives.

Hyperfols A and B: Two Highly Modified Polycyclic Polyprenylated Acylphloroglucinols from Hypericum perforatum.

Two novel polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperfols A (1) and B (2), and two known biosynthetically related precursors (3 and 4) were isolated from Hypericum perforatum. Compound 1 possesses an unprecedented 2,3-seco-PPAP with a fused 5/5/9/5 tetracyclic skeleton, and 2 features a 30-norPPAP. Their structures were established by spectroscopic analysis, computer-assisted structure elucidation software, and electronic circular dichroism calculations. Moreover, compounds 1 and 4 exhibit significant cytotoxicity against human erythroleukemia cells by inducing cell apoptosis.

Oxyresveratrol Inhibits IL-1ß-Induced Inflammation via Suppressing AKT and ERK1/2 Activation in Human Microglia, HMC3.

Oxyresveratrol (OXY), a major phytochemical component derived from several plants, has been proved to have several pharmacological properties. However, the role of OXY in regulating neuroinflammation is still unclear. Here, we focused mainly on the anti-neuroinflammatory effects at the cellular level of OXY in the interleukin-1 beta (IL-1ß)-stimulated HMC3 human microglial cell line. We demonstrated that OXY strongly decreased the release of IL-6 and MCP-1 from HMC3 cells stimulated with IL-1ß. Nevertheless, IL-1ß could not induce the secretion of TNF-α and CXCL10 in this specific cell line, and that OXY did not have any effects on reducing the basal level of these cytokines in the sample culture supernatants. The densitometry analysis of immunoreactive bands from Western blot clearly indicated that IL-1ß does not trigger the nuclear factor-kappa B (NF-κB) signaling. We discovered that OXY exerted its anti-inflammatory role in IL-1ß-induced HMC3 cells by suppressing IL-1ß-induced activation of the PI3K/AKT/p70S6K pathway. Explicitly, the presence of OXY for only 4 h could strongly inhibit AKT phosphorylation. In addition, OXY had moderate effects on inhibiting the activation of ERK1/2. Results from immunofluorescence study further confirmed that OXY inhibited the phosphorylation of AKT and ERK1/2 MAPK upon IL-1ß stimulation in individual cells. These findings suggest that the possible anti-inflammatory mechanisms of OXY in IL-1ß-induced HMC3 cells are mainly through its ability to suppress the PI3K/AKT/p70S6K and ERK1/2 MAPK signal transduction cascades. In conclusion, our study provided accumulated data that OXY is able to suppress IL-1ß stimulation signaling in human microglial cells, and we believe that OXY could be a probable pharmacologic agent for altering microglial function in the treatment of neuroinflammation.

Targeting YAP suppresses ovarian cancer progression through regulation of the PI3K/Akt/mTOR pathway.

Ovarian cancer (OC) is highly metastatic due to frequent peritoneal dissemination, and its treatment poses a major challenge in clinical practice. Yes­associated protein (YAP) is known to be associated with the development of multiple tumors. However, whether targeting YAP can restrain OC progression and the underlying mechanisms have yet to be fully elucidated. In the present study, YAP was found to be highly expressed in OC, and its expression was correlated with the prognosis of OC patients. Moreover, silencing of YAP markedly inhibited the malignant behavior of OC cells, possibly through regulation of the PI3K/Akt/mTOR pathway. Notably, peptide 17, a YAP inhibitor, exerted a significant attenuating effect on OC progression by diminishing the activation of the PI3K/Akt/mTOR pathway in vitro as well as in vivo. Taken together, these findings demonstrated that targeting YAP attenuated OC progression and suggested the potential application of peptide 17 in OC therapy, thus providing new insights into improving the treatment of OC.

Cytotoxic phenolic constituents from Hypericum japonicum.

Nine undescribed compounds, including five xanthone derivatives, two flavonoids, one 2-pyrone derivative, and one undescribed naturally occurring compound, along with 30 known phenolic compounds, were isolated from Hypericum japonicum. In addition, hyperjaponols A and B were identified as racemates. The structures and absolute configurations of the undescribed compounds were determined by comprehensive MS, NMR spectroscopy, and electronic circular dichroism (ECD) calculations. The cytotoxic effects of the isolated compounds on two human tumour cell lines (HEL and MDA-MB-231) were evaluated by the MTT assay. Eighteen compounds showed good inhibitory activities against the HEL cell line, with IC50 values of 3.53-18.7 µM, while nine compounds exhibited moderate cytotoxicity against the MDA-MB-231 cancer cell line, with IC50 values ranging from 4.92 to 10.75 µM. Their preliminary structure-activity relationship of the isolated compounds was also discussed.

Mechanical cues modulate cellular uptake of nanoparticles in cancer via clathrin-mediated and caveolae-mediated endocytosis pathways.

AIM: To investigate the influence of tissue mechanics on the cellular uptake efficiency of nanoparticles (NPs) in cancer. MATERIALS & METHODS: Collagen-coated polyacrylamide gels were prepared as model substrates. Coumarin 6-loaded poly(lactic-co-glycolic) acid micelles (C6-NPs) were prepared to investigate the cellular uptake of NPs. RESULTS: We demonstrated that substrate stiffness modulated the cellular uptake of NPs of cancer. Mechanistically, mechanical cues exerted influence on the clathrin-mediated endocytosis and caveolae-mediated endocytosis pathways, which mediated stiffness-regulated cellular uptake of NPs. CONCLUSION: Our findings shed light on the regulatory role of the mechanical cues on the cellular uptake of NPs and will facilitate the selection of clinical patients who might benefit from a given nanotherapy.

Substrate priming enhances phosphorylation by the budding yeast kinases Kin1 and Kin2.

Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

RDE: A novel approach to improve the classification performance and expressivity of KDB.

Bayesian network classifiers (BNCs) have demonstrated competitive classification performance in a variety of real-world applications. A highly scalable BNC with high expressivity is extremely desirable. This paper proposes Redundant Dependence Elimination (RDE) for improving the classification performance and expressivity of k-dependence Bayesian classifier (KDB). To demonstrate the unique characteristics of each case, RDE identifies redundant conditional dependencies and then substitute/remove them. The learned personalized k-dependence Bayesian Classifier (PKDB) can achieve high-confidence conditional probabilities, and graphically interpret the dependency relationships between attributes. Two thyroid cancer datasets and four other cancer datasets from the UCI machine learning repository are selected for our experimental study. The experimental results prove the effectiveness of the proposed algorithm in terms of zero-one loss, bias, variance and AUC.

Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library.

The human proteome encodes >500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase-substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library-kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of >100000 serine-oriented human peptides expressed heterologously in Escherichia coli, we perform in vitro kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKCß mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future.