Search

PURPOSE: This study aims to bridge the literature gap and provide a comprehensive and clear vision of managing Human Capital risk through an easy-to-apply risk management methodology. In addition, to provide an integrated process to manage Human Capital risks. METHODOLOGY: This paper conducted a mixed-method approach; risks were gathered by analyzing 14 semi-structured interviews with corporate leaders and HR expertise in 14 ICT companies in Isfahan. In the quantitative phase, variables were assessed by two methods; FMEA and Fuzzy analysis in 14 companies. FINDINGS: Results show that despite realizing the importance of human capital in achieving goals and competitiveness, companies are not ready to mitigate the Human Capital risks. Detected Risks are divided into behavioural and attitudinal risks. The Employees’ demotivation, Leaving work after receiving the required training, and Lack of commitment to the delivery time or quality are the most urgent risks that should be mitigated as prior in the ICT companies in Isfahan. ORIGINALITY: This paper is one of the few studies that holistically dealt with Human Capital risks by providing practical and managerial strategies and recommendations to mitigate the risk exposure. This paper is also one of the leading studies in introducing the FMEA method in measuring this type of risk.

To assess painful diabetic neuropathy (PDN) as a cause for refractory ear pain in type 2 diabetics.An observational prospective case series study.Otolaryngology departments of tertiary referral hospitals from November 2019 to January 2021.Sixty-eight patients with type 2 diabetes with refractory ear pain of more than 1-month duration not responding to the routine analgesics.Diagnostic intervention.The primary outcome measure was the prevalence of painful diabetic neuropathy among different causes of ear pain in the study sample of type II diabetics with refractory ear pain with an assessment of the response of these cases to routine neuropathic pain treatment with pregabalin.Fifteen out of 68 (22.1%) were diagnosed as having painful diabetic neuropathy based on the "Douleur Neuropathique en 4 Questions" (DN4) questionnaire with its mean value being 6.47±1.19. There was a highly significant improvement of the 10 items of painful diabetic neuropathy scales after 1 month of treatment ( p0.001 for all). There was a significant positive correlation between the Hemoglobin A1c level and duration of diabetes at one hand and intensity of pain derived from the painful diabetic neuropathy scale at the other hand ( p = 0.0002, and p = 0.032 respectively).Painful diabetic neuropathy showed a potential correlation with refractory ear pain in type II diabetic patients with significant improvement after painful diabetic neuropathy treatment. Further studies are needed to confirm these findings.

Purpose: To examine current decision-making preferences of Jordanian dentists when Restoring Root Filled Teeth (RFT). Materials and Methods: 834 Jordanian general dentists, prosthodontists and endodontists were invited to participate in a validated online survey with a 62% participation rate. Respondents were invited to answer 24 questions about their preferences for techniques and materials they use to restore RFT. The questions aimed at exploring restorative strategies commonly employed by Jordanian dentists when managing root filled teeth with extensive loss of tooth structure. Results: A minority of dentists consider direct resin restoration as the sole restoration for RFT with extensive loss of tooth structure. Full coverage metal-ceramic and all-ceramic crowns are more popular than endocrowns when restoring RFT with indirect restoration. Fiber posts are the most popular type of posts, followed by prefabricated metal posts. Glass ionomer is the most preferred cement for luting metal posts, while conventional dual resin cement with separate etch and rinse bonding strategy is the most employed when bonding fiber posts. The majority of dentists tend not to pretreat fiber posts. Loss of post/core retention or fracture of coronal tooth structure are the two most common reasons observed by dentists when RFT fail. Conclusion: Use of post and core is still preferred over more conservative approaches when restoring RFT with extensive loss of coronal tooth structure. Longevity of restorations can be enhanced by training practicing dentists on evidence-based adhesive strategies.

Objectives To compare the efficacy of traditional and digital communication strategies in improving compliance with fixed orthodontic therapy and to investigate the effect of gender, baseline oral hygiene habits, socioeconomics, and parents' education on orthodontic compliance. Materials and Methods Orthodontic patients were randomly allocated to three groups. Group 1 received traditional communication including verbal and written instructions, whereas group 2 and group 3 received, in addition to traditional communication, weekly text messages or e-mails with audiovisual links, respectively. Baseline demographics (age, gender, baseline oral hygiene habits, socioeconomics, and parents' education) as well as compliance indicators (treatment duration, failed appointments, incidence and total number of appliance breakages) were recorded. For statistical analysis, Pearson chi-square, independent t-test, and one-way analysis of variance were used (P < .05). Results Of 120 patients (aged 12 to 18 years) recruited, 108 completed the trial (G1 = 37, G2 = 35, G3 = 36). Weekly text messages failed to improve patient compliance. On the other hand, sending weekly e-mails with audiovisual links significantly (P = .014) reduced the incidence of appliance breakage as compared with the control group. Females had a significantly lower incidence of breakage (P = .041) and a fewer total number of breakages (P = .021). Patients from households with high income had significantly better compliance (P < .05). A higher level of parents' education was significantly associated with a lower incidence and total number of breakages (P < .01). Conclusions Communication with patients using link-rich e-mails and reminders could improve patient compliance with fixed orthodontic treatment. Female patients, high household income, and high parent education are associated with better compliance with fixed orthodontic treatment.

Background: Little prevalence studies relating to molar incisor hypomineralization (MIH) exist for Middle East populations in general and among Jordanian children in particular. Aim: To assess the prevalence and severity of MIH in a group of 3rd year class school children resident in Amman, Jordan as well as to describe its distribution in first permanent molars and incisors. Methods: A cross-sectional study with a representative sample was used. A simple random sampling technique produced 1412 school children aged 8-9- years. All children were examined by a single calibrated examiner. The first permanent molars and incisors were examined for molar incisor hypomineralization using the chart of Ghanim et al. based on the criteria of European Academy of Paediatric Dentistry (EAPD). Analysis of data was performed with a p value set at 0.05. Results: Of a total of 1412 schoolchildren 186 (13.17%) were diagnosed with MIH. Maxillary teeth were more commonly affected than the mandibular but the difference was not significant (p>0.05) and the maxillary molar was the most affected. Demarcated creamy white opacities were most frequently encountered followed by post eruptive breakdown. Atypical restorations and caries because of MIH were uncommon. More than the half of teeth was diagnosed with severe form of MIH. The severity increases with the increase of number of affected molars. Conclusion: Molar incisor hypomineralization was high among the children examined. Demarcated opacities were more frequent than breakdown. The severity of the lesions increased with the number of affected teeth.

Background Shared governance is examined through a framework for developing independent decision making in professional nursing practice and improving patient care outcomes. Aims This study is designed to obtain a baseline measurement of the degree of shared governance in a selected children’s hospital in Saudi Arabia. Methods The study was guided by the Donabedian model. The Professional Nursing Governance Index was used. A total of 400 questionnaires were distributed to nurses working at the hospital, with a response rate of 77% ( n = 307). Descriptive and inferential statistics were used for analysis. Results The results corresponded with those from nurses and managers in most subscales of the Index of Professional Nursing Governance (information, goals, resources, participation and practice). However, nurses working in the operating theatre and surgical unit have a perceived higher level of shared governance than those in critical care units and medical wards. Conclusions The results could encourage shareholders and leaders in the nursing field to develop the perception of shared governance by adopting a shared governance model, which in turn might improve the quality of nursing care .

Basma Damiri,1 Omar Khatib,2 Zaher Nazzal,3 Diala Sanduka,2 Siwar Igbaria,2 Ammar Thabaleh,2 Ahmad Farhoud,2 Lubna Saudi,3 Souad Belkebir,3 Rayyan Al Ali,4 Mohammed Alili,2 Mahmoud Hamdan,5 Omar A Safarini,2 Omar Younis2 1Medicine & Health Science Faculty, Drug, and Toxicology Division, An-Najah National University, Nablus, 00970, Palestine; 2Medicine & Health Science Faculty, Department of Medicine, An-Najah National University, Nablus, 00970, Palestine; 3Medicine & Health Science Faculty, Family and Community Medicine, An-Najah National University, Nablus, 00970, Palestine; 4Medicine & Health Sciences Faculty, Forensic Medicine Institute, An-Najah National University, Nablus, 00970, Palestine; 5Medicine & Health Science Faculty, Graduate School, Clinical Laboratory Science Program, An-Najah National University, Nablus, 00970, PalestineCorrespondence: Basma DamiriFaculty of Medicine and Health Sciences, Drug and Toxicology Division, An-Najah National University, P.O. Box 7, Nablus, 00970, PalestineTel +972592234270Email bdamiri@najah.eduIntroduction: Due to their stressful lives, Palestinian refugees are prone to use cognitive enhancers (CE) and psychostimulant substances (PS). Recognising health problems associated with CE/PS use can indicate how preventive programs should be directed towards adolescents. Unfortunately, research has not thoroughly investigated the health problems related to tobacco and caffeine products, the most socially acceptable CE/PS among Palestinian refugee adolescents.Methodology: In 2021, schoolchildren from five Palestinian refugee camps (N=271) aged 12-< 16 years agreed to give blood samples and to be interviewed to address the association between metabolic syndrome (MetS) and tobacco smoking, coffee, energy drinks (ED), black tea, and chocolate consumption. MetS was diagnosed based on International Diabetic Federation Criteria. To address the association, we used binary logistic regression models adjusted to age, gender, BMI, and central obesity.Results: The prevalence of MetS was [17 (6.3%)]; (7.4%) in males, (5.1%) in females. The prevalence of substance use was as follows: ED [163 (48.9%)], coffee [255 (76.6%)], black tea [295 (89.1%)], cigarette [35 (10.5%)], waterpipe [59 (17.7%)], and chocolate [309 (93.6%)], with male predominance. Cigarette smokers have increased central obesity (p-value=0.024), and decreased HDL-cholesterol (p-value=0.015) than non-smokers. Similar results were observed for waterpipe smokers besides the increased levels of triglycerides (p-value=0.01). ED consumers have increased central obesity (p-value=0.03) and fast blood sugar (p-value=0.003) than non-consumers. Chocolate consumers have decreased central obesity (p-value=0.008) and increased HDL levels (p-value=0.04) than non-consumers. ED consumers were significantly at higher risk of MetS (OR=9.97, p-value=0.019) than non-consumers and chocolate consumers were at lower risk of MetS (OR=0.14, p-value=0.046) than non-consumers. Waterpipe smokers were at a higher risk of having increased plasma triglycerides than non-consumers (OR=3.4, p-value=0.03).Conclusion: The high prevalence of ED consumption and waterpipe smoking in Palestinian refugee adolescents is problematic. Their use was associated with higher odds of MetS, dyslipidemia, and central obesity, which justifies greater attention.Keywords: refugee adolescents, metabolic syndrome, tobacco smoking, cognitive enhancers and psychostimulants, dyslipidemia, UNRWA

Background: Nurses are the heart of the palliative care team who provide high standards of care since they spend the longest time with patients. However, lack of adequate knowledge of palliative care is considered as one of the main obstacles to palliative care improvement. Aim: A survey was conducted to assess Saudi nursing students’ knowledge about palliative care. Methods: Using a descriptive design, 154 students who enrolled in first year and who will graduate within 1 year were surveyed in a nursing college located in Riyadh, the capital of Saudi Arabia. The Palliative Care Quiz for Nursing (PCQN) was used to assess students’ knowledge. Results: The sample mean age was 22.1 (standard deviation (SD): 2.2), and most of the students were predominately female (93; 60.4%) and the majority were senior students who will be graduated within the current academic year (105; 68.2%). The total mean score of palliative care knowledge was low at 7.30 (SD: 0.56; range: 0-13), and the number of correctly answered statements ranged from 16 (10.4%, statement no. 7) to 91 (59.1%, statement no. 18). Eleven misconceptions about palliative care among participants are conceptually categorized into 3categories: principle and philosophy of palliative care, pain and symptom management, and psychological and spiritual care. Conclusion: Integrating palliative care education within the nursing programs is the backbone to improve nursing students’ knowledge, namely, in principles of palliative care and symptom management.

BACKGROUND/AIMS: Dental trauma is a major public health problem. However, baseline data regarding traumatic injuries to primary teeth in Jordan are lacking. The study aimed at evaluating the prevalence of traumatic dental injuries to primary anterior teeth among preschool children in Amman (Jordan), investigating the relationship between dental trauma and associated factors, and assessing the treatment provided and treatment need. MATERIALS AND METHODS: After obtaining ethical approval and parental consent, a cross-sectional population-based study examined a total of 1198 children attending 39 preschools randomly selected from different areas of Amman. Chi-square test and stepwise logistic regression modeling were used for statistical analysis. RESULTS: The prevalence of traumatic dental injuries was 26.4%. The upper incisors were more likely to sustain dental trauma (91.7%). No statistically significant association was established between dental trauma and any of the socio-demographic variables. The most common type of dental trauma was enamel fracture (43.1%) followed by pulp injury (39.7%). The odds ratio suggested that the risk of dental trauma was 1.89 times greater if the overjet was >3 mm, 1.93 times greater if the child had an anterior open bite, and 2.56 times greater if the child had inadequate lip coverage. Only 25.3% of children diagnosed with a TDI visited a dentist following their trauma. CONCLUSIONS: The prevalence of dental trauma among preschool children in Amman (Jordan) was high; therefore, it is highly recommended to plan campaigns targeting parents, children, and medical/dental care providers that stress the importance of preventing dental trauma and treating it promptly. Language: en

Since stress plays a role in the onset and physiopathology of psychiatric diseases, animal models of chronic stress may offer insights into pathways operating in mood disorders. The aim of this study was to identify the molecular changes induced in rat hippocampus by repeated exposure to psychosocial stress with a proteomic technique. In the social defeat model, the experimental animal was defeated by a dominant male eight times. Additional groups of rats were submitted to a single defeat or placed in an empty cage (controls). The open field test was carried out on parallel animal groups. The day after the last exposure, levels of hippocampal proteins were compared between groups after separation by 2-D gel electrophoresis and image analysis. Spots showing significantly altered levels were submitted to peptide fingerprinting mass spectrometry for protein identification. The intensity of 69 spots was significantly modified by repeated stress and 21 proteins were unambiguously identified, belonging to different cellular functions, including protein folding, signal transduction, synaptic plasticity, cytoskeleton regulation and energy metabolism. This work identified molecular changes in protein levels caused by exposure to repeated psychosocial stress. The pattern of changes induced by repeated stress was quantitatively and qualitatively different from that observed after a single exposure. Several changed proteins have already been associated with stress-related responses; some of them are here described for the first time in relation to stress.

Neuroblastoma is the most common extracranial solid tumour of childhood and comprises up to 50% of malignancies among infants. There is a great need of designing novel therapeutic strategies and proteome analysis is one approach for defining markers useful for tumour diagnosis, as well as molecular targets for novel experimental therapies. We started by comparing healthy adrenal glands (which are the election organs developing primary neuroblastoma, NB, tumours) and adrenal glands carrying primary NB tumours, taken from nude mice. Standard maps of healthy and tumour samples were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups, were analysed by MALDI-TOF mass spectrometry and 14 of these spots were identified so far. Among these proteins, of particular interest are the down-regulated proteins adrenodoxin (21-folds), carbonic anhydrase III (eight-folds) and aldose reductase related protein I (eight-folds), as well as the up-regulated protein peptidyl-propyl cis-trans isomerase A (five-folds). Moreover new proteins, which were absent in control samples, were expressed in tumour samples, such as nucleophosmin (NPM) and stathmin (oncoprotein 18).

Summary. The purpose of this paper is to report the development of multiple odontogenic keratocysts (OKCs) in a 15-year-old female with nevoid basal cell carcinoma syndrome (NBCCS) and review the literature pertinent to NBCCS. Although more than 100 abnormalities have been reported in NBCCS, the development of OKCs is one of its principle features. In view of this, the patient was subjected to further medical, dermatological and radiographic investigation. Multiple basal cell naevi and skeletal anomalies associated with NBCCS were found. Because of the autosomal dominant inheritance of this syndrome, the patient's family was then investigated. The patient's father was found to have multiple OKCs. The report highlights the need for vigilance in considering the diagnosis of NBCCS in all cases of OKCs, particularly those affecting young patients.

Summary. Objectives. To assess dental fluorosis, provide base-line data among Jordanian children and to compare fluorosis in incisor teeth between areas with different fluoride levels. Design. Cross-sectional study among school children. Sample and methods. A sample of 1878 subjects aged 12 years, 940 boys and 938 girls, were randomly selected from 128 schools in urban (69 schools) and rural (59 schools) areas. The Thylstrup & Fejerskov (TF) index was used to record the dental fluorosis on the labial surfaces of incisors. Results. A total of 18·5% of the children examined showed dental fluorosis. The difference in fluorosis between males (17%) and females (20%) was not statistically significant (P > 0·05). Children in rural areas had a higher prevalence of fluorosis (31·8%) than their counterparts in urban areas (11·3%), a difference that was statistically significant (P

Summary. Introduction. A review of the literature relating to supernumerary teeth is presented along with a survey of 152 cases. Methods. The study population consisted of 152 children who visited the department of Paediatric Dentistry at the Jordan University Hospital. Patients ranged in age from 5 to 15 years. Supernumeraries were detected by clinical examination and radiographs. Results. Males were affected more than females with a sex ratio of 2·2 : 1. Seventy-seven percent of the patients had one supernumerary tooth, 18·4% had double teeth, and 4·6% had three or more supernumeraries. Ninety percent of the supernumerary teeth occurred in the premaxilla, of which 92·8% were in the central incisor region and of these latter 25% were located in the midline. The other 10·4% of the supernumeraries were located in the premolar, canine, molar, and lower central incisor regions. Two cases were of non-syndrome supernumerary teeth. Seventy-five percent of the supernumeraries were conical, 83·1% were in the normal vertical position and 26·5% were erupted. Conical-shaped supernumerary teeth had a significantly higher rate of eruption compared to the tuberculate type.

Summary. Objectives. The objectives of the study were: (i) to assess the level of dental knowledge and attitudes towards child dental care among parents in Jordan, (ii) to analyse the oral health care habits of schoolchildren and parents, and (iii) to evaluate the oral hygiene habits and use of professional dental services of children in relation to socio-demographic conditions. Design. Cross-sectional, self-administered questionnaires for parents. Sample and methods. National representative sample of 6–16-year-olds living in urban areas (n = 1556, children and parents, response rate 92%, 48·7% boys, 51·3% girls). Results. In all, 80% of the parents knew about the harmful effect of sugar and 79% thought that poor oral hygiene may induce dental caries. In addition to proper oral hygiene (79%) and restriction of sugar/sweets (42%), 36% of the parents emphasized regular dental visits for the prevention of dental disease in children. However, most children saw a dentist for symptomatic reasons only (86%), while 11% attended for dental check-ups. At their last visit to the dentist, 49% of the children had tooth extraction and only 8% had preventive services (fissure sealing). Toothbrushing at least twice a day was reported for 31% of the children; 14% of children aged 6–9 years had assistance from adults in brushing. Dental care habits of children were highly affected by dental visiting habits of parents, and variations by level of education of parents were also found. Conclusions. The discrepancy between dental knowledge and attitudes of parents and oral health care practices indicate the need for oral health education. School-based oral health promotion programmes should be established in Jordan to influence the oral health behaviour of children and parents and to avoid further deterioration in their oral health.

Supercritical fluid chromatography coupled to a hybrid mass spectrometer (Q-Tof2) equipped with electrospray ion source has been used to separate and characterise a wide range of pharmaceutical racemates. We have chosen diverse molecular structures to demonstrate the potential of such experimental arrangement for high throughput analyses. The use of three different chiral stationary phases and different pressure/temperature working conditions provided clear indications on how such a high throughput method can be developed. The use of mass spectrometry was found to be essential for an unambiguous assignment of the eluting components particularly in the case of complex mixtures. The direct coupling of both systems without the need for a special interface resulted in similar peak shapes and peak widths in the UV and total ion current (TIC) chromatograms.

The development of a two-dimensional (2-D) map of rat polymorphonuclear (PMN) leukocytes is here reported for the first time. The map is built up by utilizing a wide immobilized pH gradient (IPG), pH 3-10, in the first dimension and also a narrower IPG pH 4.5-8.5 gradient. In addition, the map is constructed by adopting the most recent protocols in 2-D mapping, which call for reduction and alkylation of the sample prior to the start of any electrophoretic step, including the IPG dimension. Fifty-two major protein spots have been so far identified by utilizing both matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray quadrupole (Q)-TOF mass spectrometry. A large number of house-keeping and cytoskeleton proteins were detected, together with proteins which are specific to PMN organelles or related to PMN functions such as phagocytosis and chemotaxis. The results obtained demonstrate the possibility of obtaining a single 2-D gel based proteomic map of PMN with representative proteins from different cellular compartments, also including membrane components, allowing the study of PMN protein expression on a proteome-wide scale. The aim of this project is to build an extensive database of such proteins, to be utilized for future studies where the expression of PMN proteins is used as a disease- or drug treatment marker.

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

The present review highlights some important alkylation pathways of proteins, as measured by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometric analysis, engendered by acrylamide and a number of its derivatives, including N-substituted acrylamides, cross-linkers and Immobilines (the acrylamido weak acids and bases used to create immobilized pH gradients). The present data are of relevance in two-dimensional maps and proteome analysis. It is shown that acrylamide can alkylate the -SH group of proteins even when engaged in disulfide bridges. An order of reactivity is obtained for a series of cross-linkers, which are shown to have an extremely reacting double bond, with the second one almost unreactive, originating "pendant, unreacted ends", which can subtract proteins migrating in a gel by covalently affixing them to it. An analogous reactivity scale is constructed also for the Immobiline chemicals, whose reactivity is shown to be linearly dependent on the pK values, the least reacting species being the acidic compounds. When analyzing real-life samples by two-dimensional (2-D) maps, like milk powders, a number of modifications can be detected by MALDI-TOF mass spectra of eluted spots, including variable phosphorylation sites (up to nine) and lactosyl moieties. If, for eluting such spots, formic acid is used, MALDI-TOF mass spectrometry (MS) reveals an incredible number of formylation sites, on Ser and Thr residues.

A number of cross-linkers that are commonly used in polyacrylamide gels have been incubated with bovine beta-lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization-mass spectrometry. At concentrations of 0.1, 1, and 20 mM of each cross-linker incubated for 1 h with 50 pmol/microL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N'-bisacrylylcystamine > bisacrylyl piperazine > N,N'-methylenebisacrylamide >> N,N'-diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post-source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross-linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross-linkers. Investigation of solutions containing a cross-linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.

A number of Immobilines, with pK 1.0-10.3, were incubated with two proteins, bovine alpha-lactalbumin (pI 4.80) and chicken egg lysozyme (pI 9.32), at pH approximately 9-10 and the resulting solutions were examined by matrix assisted laser desorption/ionization mass spectrometry. The reflectron mode of the same technique was also used to analyze a number of tryptic digests of some solutions. The extent and the number of detected alkylation sites associated with the acidic protein were found to be linearly proportional to the pK values of the investigated Immobilines, an effect which was less evident for the basic protein. The high resolution measurements of some tryptic digests indicate the cysteine residues as the likely sites of alkylation. The implications of the present data for isoelectric focusing separations on immobilized pH gradients and for two-dimensional maps are discussed.

Two mixtures of proteins having molecular weights in the range approximately 8-97 kDa were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and examined by delayed extraction matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). Part of our aim in this study is to gain more insight into the influence of the various experimental conditions on the overall quality of the acquired mass spectral data. Different protein extraction procedures, two staining agents, and extraction times, were among the parameters assessed. In terms of the overall quality of the acquired mass spectra and the speed of protein recovery, ultrasonic assisted passive elution, into a solvent mixture containing formic acid/acetonitrile/2-isopropanol/water, was found to be more efficient than other elution procedures. The higher resolution associated with the delayed extraction mode allowed the identification of a number of protein modifications, including multiple formylation provoked by formic acid, cysteine alkylation caused by unpolymerised acrylamide monomers, and complexation with the staining reagents. The detection of these modifications, however, was limited to proteins under 30 kDa. Analysis of a ubiquitin tryptic digest by reflectron MALDI time-of-flight (TOF) allowed reliable identification of a number of the formylation sites.

A number of proteins and peptides have been incubated with some Immobiline chemicals commonly used in the production of immobilised pH gradients for isoelectric focusing. After various incubation intervals, the resulting reaction mixtures were examined by matrix-assisted laser desorption/ionisation mass spectrometry. At pH 9–10, and after 15-h incubation time, no significant interaction was observed with the two of the investigated proteins which have no Cys residues in their sequences. On the other hand, intense multiple reaction channels were observed with sequences containing a number of Cys residues. The present measurements provide useful information on the kinetics of the reaction and its sensitivity to both the pKa of the Immobiline chemicals and the presence of Cys in the investigated sequences. Post source decay measurements on peptides with and without Cys in their sequences provided unambiguous evidence for the involvement of this residue in the reaction conducted at pH 9–10. Possible implications of some of the present deductions for isoelectric focusing separations on immobilised pH gradients are discussed. Copyright © 2000 John Wiley & Sons, Ltd.

Proteins in a commercial milk powder have been separated by two-dimensional gel electrophoresis and analysed by matrix-assisted laser desorption ionisation mass spectrometry. The mass spectrometric analyses were conducted in two steps: analysis of the intact proteins following their passive extraction into a suitable solvent mixture and analysis in reflectron mode of in situ digests of a number of gel spots. The combination of the two methods allowed a reliable identification of a number of proteins, including nine caseins as well as certain protein modifications including single/multiple phosphorylation, lactose-protein conjugates and Coomassie Brilliant Blue adducts. Analyses of the intact proteins prior to their in situ digestion contributed to a more efficient and reliable consultation of protein databases.

Delayed-extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, in both linear and reflectron modes, has been used to examine the alkylation of bovine beta-lactoglobulin-bound cysteines exposed to various molar concentrations (0.5-30 mM) of acrylamide and a number of its N-substituted monomers. These measurements were conducted at pH approximately 9.5, and showed that at 0.5 mM all monomers (except N-acryloylaminopropanol) resulted in a measurable alkylation of at least one cysteine out of five. At higher concentrations (15 mM) all investigated monomers resulted in substantial alkylation which, for some, involved all five cysteine residues. Reflectron MALDI-TOF measurements of a number of alkylated protein digests revealed that, at low molar ratios, the alkylation site is influenced by the identity of the monomer. For example acrylamide and N, N-dimethylacrylamide attacked Cys(160) as well as one of the three cysteines within the tryptic fragment [102-124], while other investigated monomers did not involve Cys(160). The implications of the present data for two-dimensional (2D) gel electrophoresis, and their eventual correlation to the toxicity of the investigated monomers, are discussed.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.

In the capillary electrophoresis of double-stranded DNA in isoelectric buffers, worsening of resolution was observed in electropherograms as a function of time passed from the preparation of the separation solution, which consisted of 0.7% hydroxypropylcellulose, HPC, Mr 10(6), diluted in 150 mM histidine buffer. The DNA standards used were: kilobase pair-ladder, Marker V and Marker VI. In order to understand what happens in the histidine-HPC solution with ageing, the absorbance spectrum (200-500 nm), the conductivity and the pH of the solutions as a function of time were monitored. Fresh His gave a distinct peak at 206 nm. For all the solutions a significant diminution in the maximum absorbance value at 206 nm was observed as a function of ageing, with the concomitant appearance of a peak at 278 nm as the solutions became older. Also the conductivity increases dramatically with the ageing of the solutions and seemed to reach a plateau after ca. 40 days. In concomitance with the conductivity increments with time, the pH of the His solution (isoelectric point, pI=7.6) grew slowly up to pH 7.9; these combined data indicated that a new species contributing to the conductivity and altering the pH was formed from the His molecule, suggesting that His degraded in time. When the dipeptide His-Gly was used instead, a similar ageing phenomenon was observed, but with much reduced kinetics. Mass spectrometry, coupled to RP-HPLC, detected, in aged His solutions, in addition to intact His, two main degradation products: a 110.1 u species and a 93.2 u compound. The mass of the former coincides with the protonated species derived from the formation of a Schiff base on the alpha-amino group of His and subsequent decarboxylation without transformation of the final Schiff base into a chetonic group (a histamine-like molecule terminating with an imino, rather than with an amino group).

There is compelling evidence to suggest that cysteine-acrylamide adduct formation is a modification experienced by proteins separated by two-dimensional (2-D) gel electrophoresis. Whether the -SH group involved in such complexation is offered by a free or initially disulphide-linked cysteine residue remains an open question. To address this question a number of proteins containing free and/or disulphide-linked cysteine (Cys) residues have been incubated with acrylamide monomer and examined by delayed extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF). These data provide strong evidence to suggest that the presence of free Cys in the investigated proteins is not the most important requirement for the observation of Cys-acrylamide adducts. Unambiguous confirmation of this deduction was obtained by analysing the tryptic digests of the same proteins by reflectron MALDI-TOF. The assignment of the adduction sites was facilitated by the mass accuracy attained for the monitored tryptic fragments and their agreement with the corresponding predicted masses reported in the Swiss-Prot database. The same data suggest that at high pH the cysteine pairing is flexible enough to allow initially S–S linked residues to complex with acrylamide. It is also plausible that the -NH2 terminal blockage so often encountered in proteins electroblotted from 2-D maps could originate from carbamylation, and might not have anything to do with alkylation by free, unreacted acrylamide in polyacrylamide gels. Copyright © 1999 John Wiley & Sons, Ltd.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.

We report here the condensation of 2-amino-6-bromopyridine with ethyl 4-chloroacetoacetate in polyphosphoric acid. In this reaction, a mixture of the four possible dihalo-4H-pyrido[1,2-a]pyrimidin-4-ones 1–4 was obtained.

Covalent and noncovalent complexes involving bovine superoxide dismutase, bovine alpha-lactalbumin and two variants, A and B, of bovine beta-lactoglobulin have been examined by delayed extraction matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray mass spectrometry. Covalent complexation with acrylamide was obtained through the interaction of these proteins with acrylamide monomers while treatment with peroxynitrite yielded complexes with NO2. Some of the complexation sites with acrylamide were reliably identified by performing tryptic digestion followed by MALDI-TOF measurements in the reflection mode. These data demonstrate that the presence of free cysteine in the investigated sequences is not a precondition for the observation of Cys-acrylamide complexes. Although the bulk of the present work is dedicated to the characterization of Cys-acrylamide adducts, the preliminary data on noncovalent complexes between two of those proteins and 8-anilinonaphthalene-1-sulfonic acid (ANS) were easily observed under electrospray conditions, while under MALDI-TOF conditions only the complex with beta-lactoglobulin B was clearly evident. The presented data demonstrate the advantage of the parallel use of both ionization techniques for this type of investigation.

A number of recent studies have demonstrated that peroxynitrite (ONOO−) can react with a host of biomolecules, particularly those containing aromatic amino acid residues, resulting in a number of modifications which have been found in association with diverse pathological conditions. Electrospray ionisation tandem mass spectrometry with and without liquid chromatographic separation has been used to examine a series of model peptides following their treatment with peroxynitrite at physiological pH. The mass spectra of sequences containing two tyrosine residues showed the formation of both mononitrated and dinitrated species, while those with phenylalanine showed no detectable nitration. Tryptic digests of two of the investigated peptides were also examined by liquid chromatography/electrospray mass spectrometry, which yielded further information on the competition for NO2 by multiple nitration sites within a given sequence. In addition, tryptic digests of the mononitrated component of sequences containing tyrosine and tryptophan indicated that mononitration was limited to tyrosine. Furthermore, some of the presented data indicate that, as well as nitrotyrosine and nitrotryptophan formation, the reaction of ONOO− can result in oxidation as well as the formation of labile adducts with NO2. Copyright © 1999 John Wiley & Sons, Ltd.

Complexation of acrylamide with bovine beta-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine-acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S-S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.

This work describes the use of multi-stage liquid chromatography–electrospray mass spectrometry to characterise four potential degradation products associated with a suspension containing a newly synthesised β-lactam antibacterial compound. Initial identification of these degradates was obtained by analytical LC in which the mobile phase contained cetyltrimethylammonium bromide as ion-pairing agent and ammonium phosphate buffer, both of which are highly involatile making them incompatible with electrospray mass spectrometry. The use of multi-stage liquid chromatography maintained the initial LC conditions necessary for the desired separation and at the same time facilitated the use of mass spectrometry detection.

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.

US11 is a small basic protein composed of 161 amino acid residues, and is among the most abundant viral proteins in cells infected with herpes simplex viruses HSV1 and HSV2. The amino acid sequence [91-121] is considered essential for the binding of this protein with RNA. Automated solid phase synthesis of this fragment resulted in a crude reaction mixture containing the desired sequence as well as a number of unknown side products. On-line liquid chromatography/electrospray mass spectrometry (LC/ES-MS) and LC/ES tandem mass spectrometry (MS/MS) allowed the identification of the separated components and furnished relevant sequencing information. The unusual sequences of the monitored components, which consist of a tandemly repeated three-amino-acid motif with the sequence Arg-X-Pro, where X is an acidic or uncharged polar amino acid residue, yielded product ion spectra lacking substantial sequence information and rich in fragment ions manifesting the neutral losses 17, 42 and 60 u.

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.

Solid-phase synthesis of (Cys)bombolitin peptide resulted in a crude reaction mixture containing the target peptide as well as a number of unknown products. The use of on-line liquid chromatography coupled to electrospray mass spectrometry (LC/ES-MS) gave a strong indication that these side-products were mainly complexes between the target peptide and mercury which was used in the form of mercury (II) acetate during the synthetic procedure. On-line LC/electrospray tandem mass spectrometry (ES-MS/MS) furnished reliable information on the amino acid sequences of both the target peptide and of the complexes with mercury or with mercury plus the b-mercaptoethanol used to eliminate the mercury at the end of the synthesis.# 1998 John Wiley & Sons, Ltd.

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimer's disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.

A series of newly synthesized peptides (M(r) = 1600-2250 Da), corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit have been examined by capillary zone electrophoresis/electrospray-mass spectrometry (CZE/ES-MS). The separation efficiency of CZE combined with the specificity of mass spectrometry allowed rapid and reliable identification of the target peptides and a number of associated side products. In solid-phase synthesis of peptides and small proteins such side products are inevitable, therefore the use of CZE and/or high-performance liquid chromatography combined with mass spectrometry is gaining a central role in such synthesis procedures.

Neuropeptide Y (NPY) and its modified form, [Leu 31 , Pro 34 ]NPY, are both thirty six amino acids long and they have relative molecular masses of 4250 and 4220. Solid-phase synthesis of both peptides resulted in complex crudes of reaction, which were investigated by means of combines high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS). The combination of these two powerful analytical techniques allowed rapid and reliable identification of the target peptides and furnished comprehensive information on other reaction products, which were mainly peptidic chains containing a smaller number of amino acids compared to those present in the intact peptides. The possible origin of such side-products and the eventual purification and unambiguous identification of both peptides are discussed.