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Cervical cancer, caused due to oncogenic types of human papillomavirus (HPV), is a leading preventable cause of cancer morbidity and mortality globally. Chronic, persistent HPV infection–induced cervical precursor lesions, if left undetected and untreated, can progress to invasive cancer. Cervical cancer screening approaches have evolved from cytology (Papanicolaou test) to highly sensitive HPV-based molecular methods and personalized, risk-stratified, management guidelines. Innovations like self-collection of samples to increase screening access, innovative triage methods to optimize management of screen positives, and scalable and efficacious precancer treatment approaches will be key to further enhance the utility of prevention interventions.

Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmaniaparasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person’s immune status with better resolution of infection.

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Affinity selection mass spectrometry (MS) or, simply, affinity mass spectrometry (AMS) is a label-free technology that has been used to identify high-affinity ligands of target proteins of interest by screening against small-molecule compound libraries and identifying molecules that are enriched in the presence of the target protein. We have previously applied Agilent Technology’s (Santa Clara, CA) RapidFire solid-phase extraction (SPE)-based high-throughput MS technology to screen small-molecule libraries using AMS. However, SPE-based technologies rely on fluidics for desalting and separation prior to mass analysis with attendant high solvent consumption, relatively high sample volume requirements, risk of sample carryover, and frequent maintenance. To address these challenges, we have established an AMS platform using a laser diode thermal desorption–atmospheric pressure chemical ionization (LDTD-APCI) ionization source (Phytronix, Quebec, Canada) coupled with a SCIEX 5600+ TripleTOF MS (Framingham, MA). We also validated a data-independent acquisition (DIA) Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) method for the robust detection and analysis of small-molecule affinity hits. An informatics platform developed in-house has resulted in a streamlined data analysis workflow for high-throughput AMS screening campaigns and reduced data processing time without compromising data quality. Finally, 68,000 compounds were screened in a single plate and affinity selected hits were confirmed in an orthogonal enzyme activity assay.

Affinity selection mass spectrometry (MS) or, simply, affinity mass spectrometry (AMS) is a label-free technology that has been used to identify high-affinity ligands of target proteins of interest by screening against small-molecule compound libraries and identifying molecules that are enriched in the presence of the target protein. We have previously applied Agilent Technology’s (Santa Clara, CA) RapidFire solid-phase extraction (SPE)-based high-throughput MS technology to screen small-molecule libraries using AMS. However, SPE-based technologies rely on fluidics for desalting and separation prior to mass analysis with attendant high solvent consumption, relatively high sample volume requirements, risk of sample carryover, and frequent maintenance. To address these challenges, we have established an AMS platform using a laser diode thermal desorption–atmospheric pressure chemical ionization (LDTD-APCI) ionization source (Phytronix, Quebec, Canada) coupled with a SCIEX 5600+ TripleTOF MS (Framingham, MA). We also validated a data-independent acquisition (DIA) Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) method for the robust detection and analysis of small-molecule affinity hits. An informatics platform developed in-house has resulted in a streamlined data analysis workflow for high-throughput AMS screening campaigns and reduced data processing time without compromising data quality. Finally, 68,000 compounds were screened in a single plate and affinity selected hits were confirmed in an orthogonal enzyme activity assay.

The two-domain actin associated protein coronin interacts with filamentous (F-) actin, facilitating diverse biological processes including cell proliferation, motility, phagocytosis, host-parasite interaction and cargo binding. The conserved N-terminal β-propeller domain is involved in protein: protein interactions, while the C-terminal coiled-coil domain mediates oligomerization, transducing conformational changes. The L. donovanicoronin coiled-coil (LdCoroCC) domain exhibited a novel topology and oligomer association with an inherent asymmetry, caused primarily by three aresidues of successive heptads. In the T.bruceihomolog (TbrCoro), two of these ‘a’residues are different (Val 493 & 507 replacing LdCoroCC Ile 486 and Met 500 respectively). The elucidated structure possesses a similar topology and assembly while comparative structural analysis shows that the T.bruceicoronin coiled-coil domain (TbrCoroCC) too possesses the asymmetry though its magnitude is smaller. Analysis identifies that the asymmetric state is stabilized via cyclic salt bridges formed by Arg 497 and Glu 504. Co-localization studies (LdCoro, TbrCoro and corresponding mutant coiled coil constructs) with actin show that there are subtle differences in their binding patterns, with the double mutant V493I–V507M showing maximal effect. None of the constructs have an effect on F-actin length. Taken together with LdCoroCC, we therefore conclude that the inherent asymmetric structures are essential for kinetoplastids, and are of interest in understanding and exploiting actin dynamics.

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Bhasmas, herbo-mineral formulations, are reported to produce adverse effects when used clinically. Therefore, the present study aimed to assess the safety of Krishna Vajra Abhraka Bhasma(KVB), through acute and subacute toxicity testing. After ethics committee approval, total 66 Wistar albino rats weighing 180-200 gms were used. Experiments were conducted as per CCSEA guidelines. In acute study, a single limit dose of 2000 mg/kg of KVBwith honey was administered to six female rats showed no mortality or adverse effects on rats' general behaviour or internal organs. Subacute toxicity studies involved 60 Wistar albino rats of either sex, randomly divided into six groups and received KVB in low (highest clinical dose extrapolated to rats), medium (double the low dose) and high (double the medium dose) dose administered orally for 28 days. The results indicated no significant differences in body and organ weights, haematological or renal function between control and treated groups. However, mid and high doses of KVB revealed reversible, dose-dependent hepatotoxicity through biochemical and histopathological evaluation, while the satellite high dose group did not exhibit this effect, confirming it as drug-induced hepatotoxicity. In conclusion, acute toxicity results positioned KVB in class V (safe) of the GHS classification. Subacute toxicity findings suggested that while low therapeutic doses were non-toxic, mid and high doses induced reversible hepatotoxicity, emphasizing the importance of careful dosage considerations and patient monitoring in Ayurvedic practice. The study contributes valuable insights into the safety profile of KVB, addressing concerns for both users and practitioners of Ayurvedic medicine.

Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone—for example, the bases participating in Watson–Crick pairing in the double helix, or the side chains contacting DNA in TALEN–DNA complexes. By contrast, specificity of protein–protein interactions usually involves backbone shape complementarity1, which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions2) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions3,4. Here we show that protein–protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations5to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli, 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro—following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing—almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.

The continuous increase in pathogenic viruses and the intensive laboratory research emphasizes the need for cost- and time-efficient drug development. This accelerates research for alternate drug candidates like antiviral peptides (AVP) that have therapeutic and prophylactic potential and gaining attention in recent times. However, diversity in their sequences, limited and non-uniform characterization often limit their applications. Isolating newer peptide backbones with required characteristics is a cumbersome process with many design–test–build cycles. Advanced deep learning approaches such as generative adversarial networks (GAN) can be helpful to expedite the initial stage of developing novel peptide drugs. In this study, we developed PandoraGAN that uses a manually curated training dataset of 130 highly active peptides that include peptides from known databases (such as AVPdb) and literature to generate novel antiviral peptides. The underlying architecture in PandoraGAN is able to learn a good representation of the implicit properties of antiviral peptides. The generated sequences from PandoraGAN are validated based on physico-chemical properties. They are also compared with the training dataset statistically using Pearson’s correlation and Mann–Whitney U-test. We, therefore, confirm that PandoraGAN is capable of generating a novel antiviral peptide backbone showing similar properties to that of the known highly active antiviral peptides. This approach exhibits a potential to discover novel patterns of AVP which may have not been seen earlier with traditional methods. To our knowledge, this is the first ever use of GAN models for antiviral peptides across the viral spectrum.

Supplemental Digital Content is available in the text

Supplemental Digital Content is available in the text.Literature published between 1975 and 2015 was systematically reviewed to conduct a case-comparator study of tissue based, immunohistochemical biomarker expression among malignant glandular histotypes of the uterine cervix so as to identify differences that could have diagnostic utility. Of the 902 abstracts, 154 articles had a full review, and 52 were included. Biomarker positivity in cases of adenocarcinoma in situ(AIS) were compared with atypical lobular endocervical glandular hyperplasia and invasive histotypes grouped as mucinous, endometrioid, adenosquamous, serous clear cell, minimal deviation-gastric type, and mesonephric carcinomas (7 AIS case-comparators). The invasive histotypes were compared with each other (30 adenocarcinoma case-comparators). Biomarker positivity in all 37 case-comparators was calculated as weighted averages of histotype-specific estimates. Unsupervised hierarchical clustering examined differences in expression and were visualized via heatmaps and dendrograms. Of the 56 biomarkers tested, 1 or more of 15 showed a 50% or more difference in positive expression in 6 (86%) of the AIS and 21 (70%) of the adenocarcinoma case-comparators. There was no data on the comparison of serous clear cell to mesonephric carcinoma. AIS case-comparator biomarkers were HIK1083, alpha SMA, PAX8, VIL1, CEA, p53, p16, and CD10, and only alpha SMA had a difference of 100%. The adenocarcinoma case-comparator biomarkers were CEA, p53, Claudin18, HIK1083, p16, Calretinin, CD10, PR, Chromogranin, MUC6, Vimentin and p63, and none had a difference of 100%. Biomarker expression in the discrimination of AIS from invasive adenocarcinoma, and the invasive histotypes from each other is understudied. One or more of 15 biomarkers could have diagnostic utility.

Background: Dysregulation of miRNAs is associated with the development of non-small cell lung cancer (NSCLC). It is imperative to study the dysregulation of miRNAs by cigarette smoke which will affect their targets, either leading to the overexpression of oncoproteins or downregulation of tumor suppressor proteins. Objective and Methods: In this study, we carried out miRNA sequencing and SILAC-based proteomic analysis of H358 cells chronically exposed to cigarette smoke condensate. Using bioinformatics analysis, we mapped the dysregulated miRNAs to differentially expressed target proteins identified in our data. Gene ontology-based enrichment and pathway analysis was performed using the deregulated targets to study the role of cigarette smoke-mediated miRNA dysregulation in NSCLC cell line. Results: miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (2 fold, Log Base 2; p-value ≤ 0.05) in H358-Smoke cells. Proteomic analysis of the smoke exposed cells compared to the untreated parental cells resulted in the quantification of 2,610 proteins, of which 690 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway study using Ingenuity Pathway Analysis (IPA) revealed activation of NRF2-mediated oxidative stress response and actin-cytoskeleton signaling, and repression of protein kinase A signaling in H358-Smoke cells. We also identified 5 novel miRNAs in H358-Smoke cells using unassigned reads of small RNA-Seq dataset. Conclusion: In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC cell line. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential therapeutic targets and/or molecular markers in NSCLC especially in smokers.

Supplemental Digital Content is available in the text.Immunohistochemistry is widely used to support a pathology diagnosis of cervical adenocarcinoma despite the absence of a systematic review and meta-analysis of the published data. This systematic review and meta-analysis was performed to investigate the sensitivity and specificity of immunohistochemistry biomarkers in the tissue-based diagnosis of cervical adenocarcinoma histotypes compared with normal endocervix and benign glandular lesions. The systematic review and meta-analysis used a PICOT framework and QUADAS-2 to evaluate the quality of included studies. The literature search spanned 40 years and ended June 30, 2015. Abstracts of identified records were independently screened by 2 of the authors who then conducted a full-text review of selected articles. Sensitivity and specificity of immunohistochemistry expression in malignant glandular lesions of the cervix classified per WHO 2003 compared with 5 benign comparators (normal/benign endocervix, and benign endocervical, endometrioid, gastric, and mesonephric lesions) were calculated. Of 902 abstracts screened, 154 articles were selected for full review. Twenty-five articles with results for 36 biomarkers were included. The only biomarker with enough studies for a meta-analysis was p16 and the definition of positive p16 staining among them was variable. Nevertheless, any positive p16 expression was sensitive, ranging from 0.94 to 0.98 with narrow confidence intervals (CIs), for adenocarcinoma in situ (AIS) and mucinous adenocarcinomas in comparison with normal/benign endocervix and benign endocervical and endometrioid lesions. Specificity for AIS and mucinous adenocarcinomas was also high with narrow CIs compared with benign endocervical lesions. The specificity was high for AIS, 0.99 (0.24, 1.0), and mucinous adenocarcinoma, 0.95 (0.52, 1.0), compared with normal/benign endocervix but with wider CIs, and low with very wide CIs compared with benign endometrioid lesions: 0.31 (0.00, 0.99) and 0.34 (0.00, 0.99), respectively. Results from single studies showed that p16, p16/Ki67 dual stain, ProExC, CEA, ESA, HIK1083, Claudin 18, and ER loss in perilesional stromal cells were useful with high (≥0.75) sensitivity and specificity estimates in ≥1 malignant versus benign comparisons. None of the biomarkers had highly useful sensitivity and specificity estimates for AIS, mucinous adenocarcinomas, or minimal deviation adenocarcinoma/gastric adenocarcinoma compared with benign gastric or mesonephric lesions or for mesonephric carcinoma compared with normal/benign endocervix, benign endocervical, endometrial, or mesonephric lesions. Any expression of p16 supports a diagnosis of AIS and mucinous adenocarcinomas in comparison with normal/benign endocervix and benign endocervical lesions. The majority of studies did not separate mosaic/focal p16 staining from diffuse staining as a distinct pattern of p16 overexpression and this may have contributed to the poor performance of p16 in distinguishing AIS and mucinous adenocarcinomas from benign endometrioid lesions. Single studies support further investigation of 8 additional biomarkers that have highly useful sensitivity and specificity estimates for ≥1 malignant glandular lesions compared with ≥1 of the 5 benign comparators.

Mechanisms by which NPM-ALK signaling regulates cell migration, invasion and contributes to the oncogenesis of anaplastic large cell lymphoma (ALCL) are not completely understood. In an attempt to identify novel actin signaling pathways regulated by NPM-ALK, a comprehensive phosphoproteome analysis of ALCL cell lines was performed in the presence or absence of NPM-ALK activity. Numerous phosphoproteins involved in actin dynamics including Wiskott–Aldrich syndrome protein (WASp) were regulated by NPM-ALK. Network analysis revealed that WASp is a central component of the NPM-ALK-dependent actin signaling pathway. Here we show that NPM-ALK phosphorylates WASp at its known activation site (Y290) as well as at a novel residue (Y102). Phosphorylation of WASp at Y102 negatively regulates its interaction with Wiskott–Aldrich interacting protein and decreases its protein stability. Phosphorylation of WASp at Y102 enhances anchorage-independent growth and tumor growth in an in vivoxenograft model and enhances invasive properties of ALCL. We show that knock-down of WASp or expression of Y102F mutant of WASp decreases colony formation and in vivotumor growth. Our results show that WASp is a novel substrate of ALK and has a critical role in regulating invasiveness and oncogenesis of ALCL.

ABSRTRACTSignaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.

Chewing tobacco is a common practice in certain socio-economic sections of southern Asia, particularly in the Indian subcontinent and has been well associated with head and neck squamous cell carcinoma. The molecular mechanisms of chewing tobacco which leads to malignancy remains unclear. In large majority of studies, short-term exposure to tobacco has been evaluated. From a biological perspective, however, long-term (chronic) exposure to tobacco mimics the pathogenesis of oral cancer more closely. We developed a cell line model to investigate the chronic effects of chewing tobacco. Chronic exposure to tobacco resulted in higher cellular proliferation and invasive ability of the normal oral keratinocytes (OKF6/TERT1). We carried out quantitative proteomic analysis of OKF6/TERT1 cells chronically treated with chewing tobacco compared to the untreated cells. We identified a total of 3,636 proteins among which expression of 408 proteins were found to be significantly altered. Among the overexpressed proteins, stearoyl-CoA desaturase (SCD) was found to be 2.6-fold overexpressed in the tobacco treated cells. Silencing/inhibition of SCD using its specific siRNA or inhibitor led to a decrease in cellular proliferation, invasion and colony forming ability of not only the tobacco treated cells but also in a panel of head and neck cancer cell lines. These findings suggest that chronic exposure to chewing tobacco induced carcinogenesis in non-malignant oral epithelial cells and SCD plays an essential role in this process. The current study provides evidence that SCD can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients who are users of tobacco.

Gastric cancer is one of the most common gastrointestinal malignancies and is associated with poor prognosis. Exploring alterations in the proteomic landscape of gastric cancer is likely to provide potential biomarkers for early detection and molecules for targeted therapeutic intervention. Using iTRAQ-based quantitative proteomic analysis, we identified 22 proteins that were overexpressed and 17 proteins that were downregulated in gastric tumor tissues as compared to the adjacent normal tissue. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was found to be 7-fold overexpressed in gastric tumor tissues. Immunohistochemical labeling of tumor tissue microarrays for validation of CAMKK2 overexpression revealed that it was indeed overexpressed in 94% (92 of 98) of gastric cancer cases. Silencing of CAMKK2using siRNA significantly reduced cell proliferation, colony formation and invasion of gastric cancer cells. Our results demonstrate that CAMKK2 signals in gastric cancer through AMPK activation and suggest that CAMKK2 could be a novel therapeutic target in gastric cancer.

The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1 (5q35) and TYK2 (19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2 fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n = 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA–mediated silencing of TYK2 abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2 translocations.

The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1(5q35) and TYK2(19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n = 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA–mediated silencing of TYK2abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2translocations.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5Bin 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RGmutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5Bhave not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5Bmutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

ABSTRACTAnal human papillomavirus (HPV) infections are common, and the incidence of anal cancer is high in HIV-infected men who have sex with men (MSM). To evaluate the performance of HPV assays in anal samples, we compared the cobas HPV test (cobas) to the Roche Linear Array HPV genotyping assay (LA) and cytology in HIV-infected MSM. Cytology and cobas and LA HPV testing were conducted for 342 subjects. We calculated agreement between the HPV assays and the clinical performance of HPV testing and HPV genotyping alone and in combination with anal cytology. We observed high agreement between cobas and LA, with cobas more likely than LA to show positive results for HPV16, HPV18, and other carcinogenic types. Specimens testing positive in cobas but not in LA were more likely to be positive for other markers of HPV-related disease compared to those testing negative in both assays, suggesting that at least some of these were true positives for HPV. cobas and LA showed high sensitivities but low specificities for the detection of anal intraepithelial neoplasia grade 2/3 (AIN2/3) in this population (100% sensitivity and 26% specificity for cobas versus 98.4% sensitivity and 28.9% specificity for LA). A combination of anal cytology and HPV genotyping provided the highest accuracy for detecting anal precancer. A higher HPV load was associated with a higher risk of AIN2/3 with HPV16 (Ptrend< 0.001), HPV18 (Ptrend= 0.07), and other carcinogenic types (Ptrend< 0.001). We demonstrate that cobas can be used for HPV detection in anal cytology specimens. Additional tests are necessary to identify men at the highest risk of anal cancer among those infected with high-risk HPV.

ABSTRACTWhile urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P= 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.

ABSTRACTLeishmania, like other eukaryotes, contains large amounts of actin and a number of actin-related and actin binding proteins. Our earlier studies have shown that deletion of the gene corresponding to Leishmaniaactin-depolymerizing protein (ADF/cofilin) adversely affects flagellum assembly, intracellular trafficking, and cell division. To further analyze this, we have now created ADF/cofilin site-specific point mutants and then examined (i) the actin-depolymerizing, G-actin binding, and actin-bound nucleotide exchange activities of the mutant proteins and (ii) the effect of overexpression of these proteins in wild-type cells. Here we show that S4D mutant protein failed to depolymerize F-actin but weakly bound G-actin and inhibited the exchange of G-actin-bound nucleotide. We further observed that overexpression of this protein impaired flagellum assembly and consequently cell motility by severely impairing the assembly of the paraflagellar rod, without significantly affecting vesicular trafficking or cell growth. Taken together, these results indicate that dynamic actin is essentially required in assembly of the eukaryotic flagellum.

Esophageal squamous cell carcinoma (ESCC) is among the top ten most frequent malignancies worldwide. In this study, our objective was to identify potential biomarkers for ESCC through a quantitative proteomic approach using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. We compared the protein expression profiles of ESCC tumor tissues with the corresponding adjacent normal tissue from ten patients. LC-MS/MS analysis of strong cation exchange chromatography fractions was carried out on an Accurate Mass QTOF mass spectrometer, which led to the identification of 687 proteins. In all, 257 proteins were identified as differentially expressed in ESCC as compared to normal. We found several previously known protein biomarkers to be upregulated in ESCC including thrombospondin 1 (THBS1), periostin 1 (POSTN) and heat shock 70 kDa protein 9 (HSPA9) confirming the validity of our approach. In addition, several novel proteins that had not been reported previously were identified in our screen. These novel biomarker candidates included prosaposin (PSAP), plectin 1 (PLEC1) and protein disulfide isomerase A 4 (PDIA4) that were further validated to be overexpressed by immunohistochemical labeling using tissue microarrays. The success of our study shows that this mass spectrometric strategy can be applied to cancers in general to develop a panel of candidate biomarkers, which can then be validated by other techniques.

The polycomb group protein BMI1 has been linked to proliferation, senescence, cancer progression and stem cell phenotype. At present, very little is known about its regulation. Here, we report that BMI1 contains a functional recognition motif for the F box protein βTrCP, which regulates ubiquitination and proteasome-mediated degradation of various proteins. We show that overexpression of wild-type βTrCP but not the ΔF mutant of it promotes BMI1 ubiquitination and degradation, and knockdown of βTrCP results in increased expression of BMI1. Furthermore, a mutant of BMI1 with an altered βTrCP recognition motif is much more stable than wild-type BMI1. We also show that wild-type BMI1 but not the mutant BMI1 interacts with βTrCP. Accordingly, compared to wild-type BMI1, mutant protein exhibited increased pro-oncogenic activity. In summary, our findings suggest that βTrCP regulates turnover of BMI1 and its function relevant to oncogenesis, cellular senescence and aging.

The identification of secreted proteins that are differentially expressed between non-neoplastic and esophageal squamous cell carcinoma (ESCC) cells can provide potential biomarkers of ESCC. We used a SILAC-based quantitative proteomic approach to compare the secretome of ESCC cells with that of non-neoplastic esophageal squamous epithelial cells. Proteins were resolved by SDS-PAGE, and tandem mass spectrometry analysis (LC-MS/MS) of in-gel trypsin-digested peptides was carried out on a high-accuracy qTOF mass spectrometer. In total, we identified 441 proteins in the combined secretomes, including 120 proteins with >2-fold upregulation in the ESCC secretome vs. that of non-neoplastic esophageal squamous epithelial cells. In this study, several potential protein biomarkers previously known to be increased in ESCC including matrix metalloproteinase 1, transferrin receptor, and transforming growth factor beta-induced 68 kDa were identified as overexpressed in the ESCC-derived secretome. In addition, we identified several novel proteins that have not been previously reported to be associated with ESCC. Among the novel candidate proteins identified, protein disulfide isomerase family a member 3 (PDIA3), GDP dissociation inhibitor 2 (GDI2), and lectin galactoside binding soluble 3 binding protein (LGALS3BP) were further validated by immunoblot analysis and immunohistochemical labeling using tissue microarrays. This tissue microarray analysis showed overexpression of protein disulfide isomerase family a member 3, GDP dissociation inhibitor 2, and lectin galactoside binding soluble 3 binding protein in 93%, 93% and 87% of 137 ESCC cases, respectively. Hence, we conclude that these potential biomarkers are excellent candidates for further evaluation to test their role and efficacy in the early detection of ESCC.

Polycomb group (PcG) proteins are overexpressed in several human malignancies including breast cancer.  In particular, aberrant expression of BMI1 and EZH2 has been linked to metastasis and poor prognosis in cancer patients. At present, very little is known about the pharmacological inhibitors of PcG proteins.  Here we show that histone deacetylase inhibitors (HDACi) downregulate expression of BMI1. Treatment of MCF10A cells, which are immortal non-transformed breast epithelial cells, and breast cancer cells with HDACi led to decreased expression of BMI1. We further show that downregulation of BMI1 by HDACi results due to the transcriptional downregulation of BMI1gene.  Specifically, we show that primary transcription and promoter activity of BMI1is suppressed upon treatment with HDACi. Furthermore, downregulation of BMI1 was accompanied by a decrease in histone 2A lysine 119 ubiquitination (H2AK119Ub), which is catalyzed by BMI1 containing polycomb repressive complex 1. HDACi treatment also led to derepression of growth inhibitory genes and putative tumor suppressors, which are known to be silenced by PcG proteins and polycomb repressive complexes (PRCs). In summary, our findings suggest that BMI1 is an important therapy target of HDACi, and that HDACi can be used alone or in combination with other therapies to inhibit growth of tumors that overexpress PcG proteins such as BMI1.

Cx3cr1CreER-driven Cre recombinase (Cre) is a widely used genetic tool for enabling gene manipulation in microglia and macrophages. However, an in-depth analysis of the possible detrimental effects of Cre activity in microglia, surprisingly, remains missing. Here, we demonstrate an age-dependent sensitivity of microglia to Cx3cr1-Cre toxicity, wherein Cre induction, specifically in early postnatal microglia, is detrimental to microglial development, proliferation, and function. Tamoxifen (TAM)-induced Cre activity leads to microglial activation, type 1 interferon (IFN-1) signaling, and increased phagocytosis, causing aberrant synaptic pruning during the early postnatal period and anxious behavior at later age. The detrimental effects of Cre induction are caused by DNA-damage-induced toxicity in microglia and are limited to the early postnatal period, showing no detrimental effects in adult microglia. Thus, our study reveals an age-dependent vulnerability of microglia to Cre activity, thereby highlighting age dependency of Cre action, which could be especially applicable in the broader context of environment-responsive cell types.

Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin‐1, a prominent NOS‐interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L‐[3H] arginine and 4,5‐diaminofluorescein diacetate (DAF‐2DA). Reverse transcriptase‐polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L‐[3H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin‐1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin‐1 in rat PMNs.

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood.

Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

The cellular immune defect in untreated Hodgkin's disease (HD) has long been recognized. This defect appears to be responsible for at least some of the morbidity and ultimately the mortality associated with the disease. In recent years, many studies have shown that the T cell component of the immune response is the apparent site where the defect in HD exists and where the immunoregulatory abnormalities that may account for the deficit are observed. The discovery of the lymphokines and monokines, comprising the human interleukin system, has elucidated some aspects of the regulatory control of the functional pathways involved in T lymphocyte activation and proliferation. The interleukin system can therefore provide the framework to dissect immunodeficiency states, such as that seen in HD. The present study indicates that HD patients' interleukin 1 (IL1) response appears to be normal, as is their T cell proliferative response to exogenous IL2. Interleukin 2 production by HD patients' peripheral blood mononuclear cells, however, is decreased when compared with age/sex-matched controls. The inability to generate IL2 after appropriate stimulation may reflect either a primary cellular defect or a regulatory defect, such as excessive immunosuppression, giving rise to the characteristic T cell hyporesponsiveness seen in HD.