Protein Ubiquitination Research in Oncology.

BACKGROUND: Ubiquitination is a vital posttranslational protein modification involved in the regulation of many eukaryotic signalling pathways. Aberrant ubiquitin signalling is known to be a molecular causality of certain cancer, neurodegenerative, immune system or cardiovascular diseases. The recent development of mass spectrometry methods enables qualitative and quantitative ubiquitination analysis in biological material from cancer patients. Research of ubiquitination may clarify the molecular cause of aberrant changes in the protein level of tumour suppressors or oncogenes. PURPOSE: We aim to explain the meaning and importance of ubiquitination in certain molecular processes taking place in the human body. We hereby emphasise the connection between ubiquitination and malignant processes. A literature search is followed by introducing our mass spectrometry platform intended for ubiquitin identification via diglycyl remnants in the CHIP protein sequence. The aim is to introduce tandem mass spectrometry identification of ubiquitin modification, ubiquitination tandem mass spectra validation and the time-dependent manner of CHIP ubiquitination to the reader. CONCLUSION: A literature search familiarises the reader with known mechanisms of aberrant ubiquitination in malignant diseases. A successfully optimised mass spectrometry platform could serve as a potent tool for determining ubiquitin position in proteins that are a part of real tumour samples.

Cooperation of Genomic, Transcriptomics and Proteomic Methods in the Detection of Mutated Proteins.

BACKGROUND: Current anti-tumour therapy is characterised by high non-specificity due to the diverse nature of tumours, which can significantly reduce its efficiency. The massive development of genomic, transcriptomic, and proteomic methods has enabled the detailed characterisation of individual tumours at the genome, transcriptome and proteome levels. Whole-genome sequencing, whole-transcriptome sequencing and exome sequencing can be listed as examples of genomics and transcriptomics methods. Those methods are suitable for detecting single-nucleotide polymorphisms. In the case of proteomic methods, where a peptide library is available, it is possible to detect mutated proteins in a bio-logical sample. Also important is software that interprets and visualises the results or facilitates conversion between data formats that are specific to the method. The combination of methods can in principle increase the likelihood of detecting new neoantigens and design-specific anti-tumour therapy. AIM: The article primarily describes the bio-informatics analysis of samples using the methods of genomics, transcriptomics and proteomics, and the possible problems which must be considered during the analysis. The article includes a description of TransPEM software designed to convert the results from the analysis of single nucleotide polymorphisms into a peptide library of sequences useful for the detection of neopeptides using proteomic methods. The publication is accompanied by a brief description of the proteomics methods using this peptide library and the summary of its limitations.

[Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines]. / Tvorba hmotnostne spektrometrických spektrálnych knizníc nádorových bunecných línií.

BACKGROUND: Cancer research often focuses on protein quantification in model cancer cell lines and cancer tissues. SWATH (sequential windowed acquisition of all theoretical fragment ion spectra), the state of the art method, enables the quantification of all proteins included in spectral library. Spectral library contains fragmentation patterns of each detectable protein in a sample. Thorough spectral library preparation will improve quantitation of low abundant proteins which usually play an important role in cancer. AIM: Our research is focused on the optimization of spectral library preparation aimed at maximizing the number of identified proteins in MCF-7 breast cancer cell line. First, we optimized the sample preparation prior entering the mass spectrometer. We examined the effects of lysis buffer composition, peptide dissolution protocol and the material of sample vial on the number of proteins identified in spectral library. Next, we optimized mass spectrometry (MS) method for spectral library data acquisition. CONCLUSION: Our thorough optimized protocol for spectral library building enabled the identification of 1,653 proteins (FDR < 1%) in 1 µg of MCF-7 lysate. This work contributed to the enhancement of protein coverage in SWATH digital biobanks which enable quantification of arbitrary protein from physically unavailable samples. In future, high quality spectral libraries could play a key role in preparing of patient proteome digital fingerprints.Key words: biomarker - mass spectrometry - proteomics - digital biobanking - SWATH - protein quantificationThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 7. 5. 2016Accepted: 9. 6. 2016.

[p SRM, SWATH and HRM -  targeted proteomics approaches on TripleTOF 5600+ mass spectrometer and their applications in oncology research]. / p SRM, SWATH a HRM -  cílené proteomické prístupy na hmotnostním spektrometru TripleTOF 5600+ a jejich aplikace v onkologickém výzkumu.

Development of novel diagnostic and therapeutic approaches in cancer research requires sensitive and quantitative assays for determination of cancer associated proteins in clinical samples. Novel quantitative targeted proteomic approaches are overviewed in this communication. A major advantage of selected reaction monitoring (SRM) and pseudo- SRM lies in the selective and sensitive quantification of selected proteins in large sample sets. As such, they represent an alternative to immunochemical approaches. On the other hand, the potential of HRM and SWATH lies in recording of digital fingerprints, which enable post acquisition quantitative proteomic data mining on a similar basis to SRM. This article shows applications of targeted proteomics in a number of cancer research studies where they were used for quantification and validation of current or potential protein bio-markers and to study their role in cancer development and progression.

Identification and characterisation of pro-metastatic targets, pathways and molecular complexes using a toolbox of proteomic technologies.

BACKGROUND: Cancer metastasis involves changes in signalling pathways, cell adhesion, migration and invasiveness. Modern proteomic, mass spectrometry based techniques enable discovery of new pro-metastatic proteins and their functional partners. Also, they might be involved in their functional characterisation and validation towards development of new diagnostic and therapeutic approaches. AIM: The aim of this communication is to describe current possibilities for proteomic techniques in the discovery and characterization of pro-metastatic targets. The NF-kappaB pathway is one of the players responsible for a number of pro-metastatic processes. The related proteins can be discovered using untargeted proteomic approaches by comparing proteomes with different metastatic potential. Stable isotope labelling based methods enable a parallel analysis of more tumour samples. The identified pro-metastatic proteins can be characterised in relationship to cell migration, invasiveness and proliferation and in terms of their involvement in molecular complexes via protein-protein interactions. Advantages of the metabolic labelling based methods can be taken in these studies, the same applies for characterisation of related surface proteins involved in cell adhesion, invasiveness and cell-to-cell communication. For clinical validation of pro-metastatic proteins in large sample cohorts, approaches of targeted proteomics based on selected reaction monitoring are becoming methods of choice. CONCLUSION: Current proteomics methods play an important role in the identification of novel pro-metastatic proteins, pathways and molecular complexes, in their functional characterisation and validation towards diagnostic and therapeutic application.

Decision-making in the colposcopy clinic--a critical analysis.

OBJECTIVE: To consider the omission of several diagnostic steps from the management of patients with high-grade squamous intraepithelial lesion (SIL) by analyzing the role of each step on the choice of treatment. STUDY DESIGN: Each diagnostic procedure was correlated to the treatment and outcome in 87 women with high-grade SIL. Treatments considered were large loop excision of the transformation zone (LLETZ) cold knife conization, and CO2 laser vaporization. RESULTS: Unsatisfactory colposcopy (P< or =0.01) and positive endocervical curettage (ECC) specimen (P< or =0.01) were essential for choice of treatment. CIN2 diagnoses of the preoperative cervical biopsy were rediagnosed as CIN3 based on the surgical specimen in 57% of the cases. The margins of 33 and 23% of surgical specimens removed by LLETZ or knife conization, respectively, displayed CIN involvement. Forty and 47% of these patients, respectively, later developed recurrent CIN. CONCLUSIONS: Omission of colposcopy and ECC could have resulted in sub-optimal treatment in many cases. Excision by LLETZ or knife conization is recommended for cases of CIN2 and CIN3. Follow up is imperative for patients with involvement of the margins.

Clinical and subclinical condyloma. Rates among male sexual partners of women with genital human papillomavirus infection.

Sixty-three regular male sexual partners of women with proven genital human papillomavirus (HPV) infection or its associated lesions were examined by colposcopy and biopsy (when necessary) to determine the prevalence of penile condyloma among them. Fifteen (24%) were found to have histologic evidence of condyloma. The severity of the women's lesions did not necessarily reflect that of lesions found in their sexual partners. The majority (54%) of the HPV-associated lesions were located on the penile body, followed by the penile root (25%). We found no cases of premalignant or malignant penile lesions. After controlling for the confounding factors of socioeconomic status and lesion location and comparing the data to those compiled from the English-language literature, we arrived at various theories about the very low rate of infection in our population. One possibility is the protective effect of circumcision, partly because the preputium is a major site of bacterial and viral colonization. The possibility of different strains of HPV in the Jewish Israeli population and certain socioeconomic factors that may limit the spread of sexually transmitted diseases may be the basis for future study.

Penile condylomata: a gynecological epidemic disease: a review of the current approach and management aspects.

Viral venereal infection caused by human Papillomavirus has reached epidemic state. The proper management of this infection in men is of great benefit, because it may possibly decrease the reservoir of disease in both sexes from which genital condylomata and associated lesions may arise. We report a selection of current knowledge about the epidemiology, etiology, diagnosis, and treatment of male condyloma, occurring predominantly among male consorts of women with genital human Papillomavirus infection. In a review of 1455 affected women, compiled from the literature, 1019 (70 per cent) of their sexual partners were diagnosed as having been infected with the same viral disease. The current diagnostic and treatment modalities and their outcomes are discussed with appropriate recommendations for their use.

Soluble interleukin-2 receptor and interleukin-2 in human amniotic fluid of normal and abnormal pregnancies.

In the present study, 63 specimens of human amniotic fluid were tested for the presence of free soluble interleukin-2 receptor (IL-2R). Thirty-two of these were also tested for the presence of IL-2. Significant reduction in free soluble IL-2R (IU/ml) or free IL-2R (IU/mg albumin) levels were found in the amniotic fluid obtained from pregnant women with Down's syndrome fetuses as compared with normal pregnancies. In addition normal amniotic fluid was found to contain low levels of IL-2, while no IL-2 was found in amniotic fluid from pregnant women with Down's syndrome fetuses when tested by two different tests.

Nasal carriage of Staphylococcus aureus: correlation with hormonal status in women.

In view of recent observations on hormone-microorganism interactions, a study of Staphylococcus aureus nasal carriage in relation to sex-hormone status was undertaken. Prospectively in 479 women attending a colpocytologic clinic, hormonal status was assessed by determining the karyopyknotic index (KI) on smears stained by the Papanicolaou method. Rates of S. aureus nasal carriage were 29.3% in premenopausal women and 21.9% in postmenopausal women (P not significant). Carriage rates were significantly higher (P = .026, chi 2 7.32) for women with high KIs (40.7%) than for those with intermediate and low KIs (27.03% and 25.1%, respectively). S. aureus nasal carriage also correlated independently and significantly with previous antibiotic use and the presence of insulin-treated diabetes mellitus. This preliminary observation confirms an association between levels of sex hormones as reflected by the KI and S. aureus nasal carriage rates.

Immunosuppressive activity of human amniotic fluid of normal and abnormal pregnancies.

Twenty specimens of amniotic fluid (AF) obtained between week 16 and 18 of gestation from normal pregnant women and six specimens from pregnant women in which trisomia of chromosome 21 was found were tested for immunosuppressive activity. Incubation of normal human donor lymphocytes with 0.2-1 mL of AF from normal pregnant women for one hour at 37 degrees C was sufficient for induction of significant inhibition of the ability of these cells to induce a local xenogeneic graft-versus-host reaction (GVHR) as well as inhibition of E and E-active rosette formation, the GVHR being the most sensitive test. On the other hand, amniotic fluid obtained from the six pregnant women in which trisomia of chromosome 21 was found showed no inhibitory activity in either the E or E-active rosette formation, nor in the local xenogeneic graft-versus-host reaction. AF from all the women tested was found to have no effect on phenotype expression of the lymphocytes, as tested by the monoclonal antibodies OKT4+ and OKT8+, nor on B-lymphocytes, as tested by surface immunoglobulins. No correlation was found between the alpha-fetoprotein levels in the sera of those women and the immunosuppressive activity. These findings indicate that genetic defects of the conceptus are not limited to the embryo but may affect the composition of immunosuppressive components present in normal amniotic fluid.

Intra-amniotic injection of oxytetracycline hydrochloride for termination of mid-trimester pregnancy.

Seventy-eight women underwent induced mid-trimester abortion. Fifty-two women aborted after an intra-amniotic injection of 1 g oxytetracycline hydrochloride. The control group comprised 16 women who received an intra-amniotic injection of hypertonic saline and 9 women with prostaglandin F2 alpha. All but 2 women aborted after one injection. Thirty-five women of the oxytetracycline hydrochloride group received intravenous oxytocin after the appearance of uterine contractions, 17 did not. The mean injection abortion interval in the women who received intravenous oxytocin was 38.6 +/- 2.7 h, whereas in the group without oxytocin it was 31.3 +/- 2.3 h. There is no statistically significant difference between these two groups (p less than 0.2). The mean injection abortion interval in the hypertonic saline group was 18.4 +/- 2.2 h. In the F2 alpha group it was 13.2 +/- 1.3 h. There is no statistically significant difference between hypertonic saline and prostaglandins (p less than 0.2). The mean injection abortion interval is significantly shorter in the F2 alpha and hypertonic saline groups as compared to the oxytetracycline hydrochloride group (p less than 0.001). It is advisable therefore to use oxytetracycline hydrochloride only in cases when the use of F2 alpha or hypertonic saline is contraindicated.

Removal of completely perforating IUDs by explorative laparotomy.

Nine patients with completely perforating intrauterine contraceptive devices are presented. In all cases, the device was removed by explorative laparotomy. In eight out of the nine women, the device was found to be embedded in the omentum or vesical peritoneal plica. The advantages of explorative laparotomy over laparoscopy or colpotomy are emphasized.

PIP: The use of laparotomy for removal of completely perforating IUDs was reviewed. A table summarizes the clinical details of 9 patients who sought removal of completely perforating IUDs at the Carmel Medical Center, Haifa, Israel, between 1979-83. The patients varied in age between 23-46 years. Only 2 of the 9 patients had reported pain during the IUD insertion. Predisposing factors that may explain the perforation were found in patient 3 (cesarean section), patient 4 (grand multipara), and patients 6, 7, and 8 (6-10 weeks postpartum). 5 of the IUDs were Lippes loops; the others were Cu-7s. All patients were asymptomatic. In 6 cases, the IUDs were deeply embedded in the omentum; in 2 cases the IUDs were found free in the posterior cul de sac. In all cases in which the IUDs were embedded, the removal was difficult and required fine dissection of the adhesions between the device and the surrounding tissues. As the routine investigations failed to show whether the perforating IUDs were embedded or free in the abdominal cavity, it is advisable to remove such devices by explorative laparotomy.

The "forgotten" intrauterine device.

It is essential to ascertain that women who had an IUD inserted at some time have indeed had the device removed. It is also important to ensure that the whole IUD was removed and that no parts were left in the uterine cavity. This is especially true in cases of infertility and should be ascertained before invasive investigations such as hysterosalpingography or laparoscopy commence. An accurate history and ultrasonography can easily rule out a forgotten or fragmented IUD, thereby preventing considerable unnecessary suffering and expense.

PIP: This article presents 3 cases that illustrate a generally overlooked complication of IUD use: a device that is forgotten by the patient and gynecologist and remains in the uterus. The cases demonstrate the need to ensure that women who have had an IUD inserted at some point have indeed had the device removed. It is especially important to ensure that there is no IUD in utero in the following 2 situations: 1) women who claim or were told the IUD fell out or in cases where the suspected spontaneous loss of an IUD was not documented radiographically; and 2) women referred for infertility investigation, who previously had an IUD and in whom removal of an intact device is not documented by a physician's report or gross pathology report. An accurate history and ultrasonography can easily rule out a forgotten or fragmented IUD, thereby preventing considerable unnecessary testing and expense.

Maternal permanent bradycardia: pregnancy and delivery.

Presented are six women with permanent bradycardia without myocardial involvement. Four women were asymptomatic, and two had repeated Adams-Stokes attacks and required the use of permanent pacemakers. All six women had 18 pregnancies with 15 deliveries of normal infants. During pregnancy and delivery all patients remained asymptomatic (functional capacity 1). The various cardiologic and obstetric aspects of these patients are discussed, and it is concluded that pregnancy and delivery in women with permanent bradycardia is possible, and their outcome is no different from that of normal, healthy women. However, cases with associated myocardial disease carry an increased risk and should therefore be carefully monitored during pregnancy and delivery.

Congenital uterine malformations as indication for cervical suture (cerclage) in habitual abortion and premature delivery.

Fifteen women with diminished fertility as a result of congenital uterine anomalies were treated by cervical suture (cerclage) in the 11th-12th week of pregnancy without surgical correction of the uterine anomaly. None of these women had either clinical or radiological evidence of cervical incompetence. As a result of the treatment, 13 of the women delivered live full-term mature infants. None of the women aborted, only two women delivered prematurely, and both infants were alive and well. Comparison of these good results to the very poor outcome of previous pregnancies in these women points to a distinct advantage to be gained by cervical suture. As a result of this experience, we recommend cervical suture in cases of diminished fertility as a result of congenital uterine anomaly. We recommend that the suture be performed before considering surgical repair of the anomaly and irrespective of lack of evidence of cervical incompetence. Only in cases where cervical suture is unsuccessful, would we recommend surgical correction according to the accepted techniques.

Aeromedical evacuation in Israel--a study of 884 cases.

We analyzed 884 moderately severe and severe casualties evacuated by the aeromedical evacuation unit of the Israel Air Force. Of these, 452 were evacuated during the "Yom Kippur" War in 1973, and 432 during 1974-76. The aeromedical teams performed 24 rescusitations and 13 emergency operations, and provided primary medical care for 37 cases; 95% of the casualties reached the next stage of evacuation without deterioration in their medical condition. These data support our concept that the participation of a flight surgeon in aeromedical evacuation is highly valuable. The advantages of small helicopters for aeromedical evacuation purposes are outlined. The suitability of the aeromedical evacuation system in Israel for wartime as well as for peacetime is pointed out.