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Introduction Acute supralevator abscess (SLA) is an uncommon and complicated form of anorectal sepsis. Its correct management is crucial to avoid complex iatrogenic fistula formation. A comprehensive review of the literature was conducted to determine the incidence of SLA and the surgical outcome with particular reference to the direction of drainage. Methods A search of Medline, PubMed and the Cochrane Library was performed to identify all studies reporting surgical drainage of SLA. Findings A total of 19 studies were identified, including 563 patients. The only two prospective studies reported an incidence of SLA of 10% and 3% in 68 and 100 patients, respectively, with anorectal sepsis. In 17 retrospective studies, the incidence ranged from 0% to 28%. Magnetic resonance imaging (MRI) was performed routinely in only one study. The surgical anatomical classification of the abscess was described in six studies diagnosed at surgery. The direction of surgical drainage whether ‘inwards’ (into the lumen) or ‘outwards’ (into the ischioanal fossa) was stated in only six studies. In two of these, the direction of drainage was contradictory to the recommendation made by Parks et al. Recurrent sepsis was reported in eight studies and ranged from 0% to 53%. Conclusions Detailed and prospective data on acute SLA are lacking. Its real incidence is unclear and it is not possible to analyse surgical outcomes conclusively according to different direction of drainage. The routine use of MRI in complicated anorectal sepsis would specify the surgical anatomy of SLA before any drainage is carried out.

We analyze an important but little‐studied institution for balancing supply risk in the management of procurement operations: performance bonding. By adding the surety as a third party that guarantees contract fulfillment between supplier and buyer, performance bonding aims to streamline the purchasing process by influencing both contractor selection in the bidding phase and contract enforcement during project execution. Using the data on US government procurement from 2005 to 2015 and exploiting an exogenous variation in the threshold for its application to construction contracts, we find that performance bonding improves contract outcomes by 10.5% and 3.7% in terms of delays and extra costs, respectively. Net of bond premia, which by law are included in the award amounts, this effect translates into savings of about 4% in the budget for federal construction projects and 16% for mid‐size projects. We provide suggestive evidence on the effectiveness of selection and monitoring by sureties as driving channels.

The impact of transanal local excision (TAE) of early rectal cancer (ERC) on subsequent completion rectal resection (CRR) for unfavorable histology or margin involvement is unclear. The aim of this study was to provide a comprehensive review of the literature on the impact of TAE on CRR in patients without neoadjuvant chemoradiotherapy (CRT). We performed a systematic review of the literature up to March 2020. Medline and Cochrane libraries were searched for studies reporting outcomes of CRR after TAE for ERC. We excluded patients who had neoadjuvant CRT and endoscopic local excision. Surgical, functional, pathological and oncological outcomes were assessed. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines were followed. Sixteen studies involving 353 patients were included. Pathology following TAE was as follows T0 = 2 (0.5%); T1 = 154 (44.7%); T2 = 142 (41.2%); T3 = 43 (12.5%); Tx = 3 (0.8%); T not reported = 9. Fifty-three percent were > T1. Abdominoperineal resection (APR) was performed in 80 (23.2%) patients. Postoperative major morbidity and mortality occurred in 22 (11.4%) and 3 (1.1%), patients, respectively. An incomplete mesorectal fascia resulting in defects of the mesorectum was reported in 30 (24.6%) cases. Thirteen (12%) patients developed recurrence: 8 (3.1%) local, 19 (7.3%) distant, 4 (1.5%) local and distant. The 5-year cancer-specific survival was 92%. Only 1 study assessed anal function reporting no continence disorders in 11 patients. In the meta-analysis, CRR after TAE showed an increased APR rate (OR 5.25; 95% CI 1.27–21.8; p 0.020) and incomplete mesorectum rate (OR 3.48; 95% CI 1.32–9.19; p 0.010) compared to primary total mesorectal excision (TME). Two case matched studies reported no difference in recurrence rate and disease free survival respectively. The data are incomplete and of low quality. There was a tendency towards an increased risk of APR and poor specimen quality. It is necessary to improve the accuracy of preoperative staging of malignant rectal tumors in patients scheduled for TAE.

Background: Gallbladder cancer (GBC) is an aggressive, uncommon malignancy, with variation in operative approaches adopted across centres and few large-scale studies to guide practice. We aimed to identify the extent of heterogeneity in GBC internationally to better inform the need for future multicentre studies. Methods: A 34-question online survey was disseminated to members of the European-African Hepatopancreatobiliary Association (EAHPBA), American Hepatopancreatobiliary Association (AHPBA) and Asia–Pacific Hepatopancreatobiliary Association (A-PHPBA) regarding practices around diagnostic workup, operative approach, utilization of neoadjuvant and adjuvant therapies and surveillance strategies. Results: Two hundred and three surgeons responded from 51 countries. High liver resection volume units (>50 resections/year) organised HPB multidisciplinary team discussion of GBCs more commonly than those with low volumes (p < 0.0001). Management practices exhibited areas of heterogeneity, particularly around operative extent. Contrary to consensus guidelines, anatomical liver resections were favoured over non-anatomical resections for T3 tumours and above, lymphadenectomy extent was lower than recommended, and a minority of respondents still routinely excised the common bile duct or port sites. Conclusion: Our findings suggest some similarities in the management of GBC internationally, but also specific areas of practice which differed from published guidelines. Transcontinental collaborative studies on GBC are necessary to establish evidence-based practice to minimise variation and optimise outcomes.

Conflict and mass displacement into Protection of Civilian (POCs) sites in South Sudan led to the breakdown of community and family structures, increasing women and girls' vulnerability to gender-based violence and exacerbating already poor sexual and reproductive health outcomes. As one component of a study on post-abortion care, this study explores community perceptions of unintended pregnancy and abortion in a POC in Juba. Four focus group discussions were conducted with 36 women and married men aged 18-45 living in the POC. Although initial reactions to induced abortion were generally negative, participants discussed that unintended pregnancy and induced abortion appeared to have increased during the current conflict. Their discussion of abortion became less condemnatory as they described changes in people's situation due to war, including instability and poverty, transactional sex, disruption of marital norms, rape, and low contraceptive use. This is one of the first studies to investigate community perceptions and practices related to unintended pregnancy and abortion in South Sudan. Despite the beliefs that these are taboo topics, the discussions provide an opening to reduce abortion stigma. To ensure lasting stigma reduction, investment in women and girls to improve gender equity is needed.

A well-functioning bureaucracy is a precondition for efficient public goods provision. However, bureaucratic decision-making is still largely seen as a black box. We provide novel insights into the preferences of bureaucrats regarding their work outcomes. We focus on a major public sector activity and survey more than 900 real-life procurement officials in Finland and Germany. The questionnaire includes hypothetical choice experiments to study the relative importance of multiple features in tender outcomes. First, bureaucrats state to have substantial discretion at work but no important incentives. Second, our experimental results show that procurers are particularly worried about avoiding negative risks concerning prices and supplier reputation. Third, an avoidance of bidders with prior bad performance appears to be an extremely important factor. Fourth, procurers value a certain degree of competition, while litigation concerns and regional favoritism play only a small role. The striking lack of heterogeneous effects points towards the role of intrinsic motivation among public buyers in countries with high public sector capacity.

Background Unsafe abortion is a leading cause of maternal mortality. In Afghanistan, which has experienced decades of armed conflict and where abortion is highly restricted, maternal mortality is high at 638 maternal deaths per 100,000 live births. Post-abortion care (PAC) is a lifesaving package of interventions to reduce morbidity and mortality related to induced or spontaneous abortion, but is rarely provided and often of poor quality, particularly in humanitarian settings. In July 2018, we conducted a study to identify the factors that influence access to and use of PAC services at Sharana Provincial Hospital. Methods In-depth interviews (IDIs) were conducted with ten women who had received PAC services at Sharana Hospital, and eight focus group discussions (FGDs) were conducted with 40 married women and 40 married men aged 18–45 from four villages surrounding Sharana Hospital. Results PAC clients and community participants discussed similar barriers to seeking PAC, including cost, distance to the health facility, the need for male accompaniment to seek care, perceived and actual quality of care, stigma and shame. Despite the mentioned stigma around abortion, community members expressed willingness to help women to receive PAC. Conclusions Our results suggest that while some barriers are not unique to PAC, others, especially those related to stigma around abortion, may be specific to PAC. It is important for the Ministry of Public Health and its partners to prioritize addressing these barriers to ensure that women have access to this critical life-saving care.

To what extent does a more competent public bureaucracy contribute to better economic outcomes? We address this question in the context of the US federal procurement of services and works, by combining contract-level data on procurement performance and bureau-level data on competence and workforce characteristics. We use the death occurrences of specific types of employees as instruments and find that an increase in bureau competence causes a significant and economically important reduction in (a) time delays, (b) cost overruns, and (c) number of renegotiations. Cooperation within the office appears to be a key driver of the findings. (JEL D73, H11, H57, L26).

Background: The incidence of Paediatric type 2 diabetes is increasing, especially in areas of deprivation. Aim: To describe the cohort of CYP with T2D in Royal London Hospital over the period 2009-2018. Methods: Retrospective analysis of patient cohort. Results: Number of new patients doubled from 2.6/year in 2009-2013 to 5.3/year in 2014-2018. Prevalence in our cohort is 7.5% (national average of 2.5%, NPDA 2017-2018). Fourty patients (25 female, 15 male) were diagnosed in 2009-2018, with a mean age at diagnosis of 13.9+/-1.7 yrs. Males had more frequently learning difficulties compared to females (40% vs 20%). Sixty % of patients were Asian compared to 28% in our T1D cohort. BMI at presentation was 31.5 kg/m2 (23 females) and 33.85 kg/m2 (13 males). BMI remained stable for females for the first year after diagnosis but in males increased to 34.6 kg/m2 (n=10, non significant (ns)). At diagnosis, Metformin was started in 38/40 patients although 7 patients reduced the dose and 6 stopped due to side effects. 12/36 patients started also on long-acting insulin (0.28+/-0.17U/kg), in 6 combined with prandial insulin (0.42+/-0.20U/kg). Seven patients started long-acting insulin at a later stage and 6 required prandial insulin too. 1 patient was treated with Sitagliptin. HbA1c at diagnosis (n=27) was 75.2+/- 20mmol/mol, similar for males and females.HbA1c dropped to 55.0+/-17.4mmol/mol after 3 months (P Nineteen of 38 patients achieved a HbA1c < 48 at least once, but only 9 of 35 achieved an HbA1c < 48 for a year. Of these, 3 continued to be on insulin and in 1 patient insulin was stopped. Two patients relapsed. Complications were as follows: 11/21 hypertension, 6/28 sleep apnoea, 10/30 raised ALT and 9/24 fatty liver. Conclusion: Learning difficulties in patients with T2D are frequent. Complications of obesity/T2D are common in this cohort. Current treatment does not achieve permanent reduction in BMI and HbA1c in most patients although temporary reduction of HbA1c is possible. New treatment approaches are needed to improve outcomes.

To what extent does a more competent public bureaucracy contribute to better eco- nomic outcomes? We address this question in the context of the US federal procurement of services and works, by combining contract-level data on procurement performance and bureau-level data on competence and workforce characteristics. We use the death occurrences of specific types of employees as instruments and find that an increase in bureau competence causes a significant and economically important reduction in: i) time delays, ii) cost overruns, and iii) number of renegotiations. Cooperation within the office appears to be a key driver of the findings.

To what extent does a more competent public bureaucracy contribute to better economic outcomes? We address this question in the context of the US federal procurement of services and works, by combining contract-level data on procurement performance and bureau-level data on competence and workforce characteristics. We use the death occurrences of specific types of employees as instruments and find that an increase in bureau competence causes a significant and economically important reduction in: i) time delays, ii) cost overruns, and iii) number of renegotiations. Cooperation within the office appears to be a key driver of the findings.

We empirically investigate the effect of procurement oversight on contract outcomes. In particular, we stress a distinction between public and private oversight: the former is a set of bureaucratic checks enacted by contracting offices, while the latter is carried out by private insurance companies whose money is at stake through the socalled performance bonding. By focusing on the U.S. federal service contracts in the period 2005-2015, we exploit an exogenous variation in the threshold for the application of both sources of oversight in order to separately estimate their causal effects on execution costs and time. We find that: (i) private oversight has a positive effect on outcomes through the screening of bidders that alters the pool of winning firms; (ii) public oversight negatively affects outcomes, due to excessive red tape induced by low-competence buyers.

There is significant evidence to show that aging is characterized by a stochastic accumulation of molecular damage and by a progressive failure of maintenance and repair processes. Protective mechanisms exist in the brain which are controlled by vitagenes and include members of the heat shock system, heme oxygenase-I, and Hsp70 as critical determinants of brain stress tolerance. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders, and the present study reports that treatment for 4 months of senescent rats with acetyl-L-carnitine induces heme oxygenase-1 as well as Hsp70 and SOD-2. This effect was associated with upregulation of GSH levels, prevention of age-related changes in mitochondrial respiratory chain complex expression, and decrease in protein carbonyls and HNE formation. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Particularly, modulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.

Efficient functioning of maintenance and repair processes seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of several genes termed "vitagenes," among these, the heat shock system, a highly conserved mechanism responsible for the preservation and repair of cellular macromolecules, such as proteins, RNAs, and DNA. Recent studies have shown that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human diseases, including ischemia and reperfusion damage, inflammation, cancer, as well as metabolic and neurodegenerative disorders. Recently, the involvement of the heme oxygenase (HO) pathway in antidegenerative mechanisms has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein. HO induction occurs together with the induction of other heat shock proteins during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms, via the heat shock response, through nutritional antioxidants or pharmacological compounds, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistently, by maintaining or recovering the activity of vitagenes, it is feasible to delay the aging process and decrease the occurrence of age-related diseases with resulting prolongation of a healthy life span.

In this work we studied the morphology of plasmas induced by single and double laser pulses, with the purpose to improve the understanding of the formation and evolution of the plume in the two cases and the reasons of the increase of sensitivity and detection limits in the double pulse configuration. Single and double pulse laser-induced breakdown spectroscopy experiments were carried out on a brass sample in air. Spectrally, temporally and spatially resolved measurements have been performed on the plume and a deconvolution algorithm has been applied on the line-of-sight integrated spectra in order to separate the spectral information coming from different regions of the plasma. While in single pulse configuration only the major elements in the matrix were observed, in double pulse case also the minor elements emission lines were measurable. The values of line emission intensity, plasma temperature and electron density have been evaluated at different positions in the plume in both the configurations. Appreciable differences in plume dimensions (three times wider in double pulse configuration), and electron density values (two times higher in single pulse case), have been found while the maximum temperature in the plasma core was similar in the two configurations. Beside the spectroscopic measurements, we used the shadowgraphic technique to observe the evolution of the plume in single and double pulse configuration. In double pulse configuration, the expansion of the plume induced by the second pulse is sensibly faster than the one induced by the first pulse and, in double pulse case, the plume tends to fill the region encompassed by the shock wave formed by the first pulse; on the other hand no shock wave produced by the second pulse was visible. The results obtained by the two different approaches were compared and an explanation of the physical processes occurring is proposed.

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Oxidative damage plays a crucial role in the brain aging process and induction of Hsps is critically utilized by brain cells in the repair process following various pathogenic insults. In the present study, we investigated, in rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant redox status. In the brain expression of Hsp72 and Hsc70 increased with age up to 28 months; at this age the maximum induction was observed in the hippocampus and substantia nigra followed by cerebellum, cortex, septum and striatum. Hsps induction was associated with significant changes in glutathione (GSH) redox state and HNE levels. Interestingly, a significant positive correlation between decrease in GSH and increase in Hsp72 was observed in all brain regions examined during aging. Analysis of mitochondrial complexes showed a progressive decrease of Complex I activity and mRNA expression in the hippocampus and a significant decrease of Complex I and IV activities in the substantia nigra and septum. Our results sustain a role for GSH redox state in Hsp expression. Increase of Hsp expression promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage. Conceivably, heat shock signal pathway by increasing cellular stress resistance may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders.

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Alzheimer's disease (AD) is a progressive disorder with cognitive and memory decline, speech loss, personality changes and synapse loss. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis. Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress.Recently, the involvement of the heme oxygenase (HO) pathway in anti-degenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein (APP). HO induction occurs together with the induction of other HSPs during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. Increasing interest has been focused on identifying dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiological events underlying AD pathology. Alzheimer's disease, in fact, involves a chronic inflammatory response associated with both brain injury and beta-amyloid associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD. Here we review the importance of the heme oxygenase pathway in brain stress tolerance and its significance as an antidegenerative mechanism potentially important in AD pathogenesis. These findings have offered new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms such as the heat shock response, through nutritional antioxidants or pharmacological compounds, represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistent with this notion, maintenance or recovery of the activity of vitagenes, such as the HO gene, conceivably may delay the aging process and decrease the occurrence of age-related neurodegenerative diseases.

Heme oxygenase-1 (HO-1) is a redox-sensitive inducible protein that provides efficient cytoprotection against oxidative stress. Curcumin, a polyphenolic natural compound that possesses anti-tumor and anti-inflammatory properties, has been reported recently to induce potently HO-1 expression in vascular endothelial cells (Free Rad Biol Med 28:1303-1312, 2000). Here, we extend our previous findings by showing that caffeic acid phenethyl ester (CAPE), another plant-derived phenolic agent, markedly increases heme oxygenase activity and HO-1 protein in astrocytes. The effect seems to be related to the peculiar chemical structures of curcumin and CAPE, because analogous antioxidants containing only portions of these two molecules were totally ineffective. At a final concentration of 30 microM, both curcumin and CAPE maximally up-regulated heme oxygenase activity while promoting marked cytotoxicity at higher concentrations (50-100 microM). Similar results were obtained with Curcumin-95, a mixture of curcuminoids commonly used as a dietary supplement. Incubation of astrocytes with curcumin or CAPE at concentrations that promoted maximal heme oxygenase activity resulted in an early increase in reduced glutathione followed by a significant elevation in oxidized glutathione contents. A curcumin-mediated increase in heme oxygenase activity was not affected by the glutathione precursor and thiol donor N-acetyl-L-cysteine. These data suggest that regulation of HO-1 expression by polyphenolic compounds is evoked by a distinctive mechanism which is not necessarily linked to changes in glutathione but might depend on redox signals sustained by specific and targeted sulfydryl groups. This study identifies a novel class of natural substances that could be used for therapeutic purposes as potent inducers of HO-1 in the protection of tissues against inflammatory and neurodegenerative conditions.

The glial fibrillary acidic protein (GFAP) is an intermediate filament protein, specific of the cytoskeleton of astrocytes in the central nervous system. In the present work, as a preliminary step to the study of glial-specific gene expression, we cloned the rat GFAP gene, and we report the sequence of 1.9 kb of the 5' flanking region, exon 1, and the majority of the first intron. By digestion with methylation-sensitive restriction enzymes followed by Southern blot analysis, the methylation status of various CpG sites was examined in this genomic segment. We tested whether structural modification of the GFAP gene, such as DNA methylation, could be related to its tissue-specific transcriptional activity. Therefore, we compared a GFAP-expressing cell population (primary culture of astroglial cells), a mixed population of GFAP-expressing and -nonexpressing cells (adult rat cerebral hemispheres), and a GFAP-nonexpressing tissue (liver). In the 5' flanking region we identified a CpG site at position -1176 whose level of methylation is inversely correlated to GFAP expression. In primary cultured astrocytes, 75% of the GFAP gene alleles were demethylated at this site, while the corresponding value obtained for the cerebral hemispheres was 45%, and for liver only 9%. On the basis of the sequence data, a CpG-rich region (putative CpG island) was identified extending from -38 to +347 and overlapping 80% of the first exon. HhaI and HpaII sites located in the putative CpG island showed a relatively high level of methylation in all the cell populations examined, and did not show any clear correlation with the level of GFAP gene expression or with the methylation status of the -1176 site. Further in vivo developmental studies and in vitro differentiation studies are necessary to better understand the functional differences of the various methylatable CpG sites in the 5' end of the GFAP gene.

Decreased expression and/or activity of antioxidant proteins leads to oxidative stress, accelerated aging, and neurodegeneration. While overwhelming levels and uncontrolled/dysregulated actions of reactive oxygen species (ROS) lead to deleterious effects, tighter regulation of those plays an important role in cell signaling. Mutations causing protein misfolding and the overload of toxic products derived from the free radical oxidation of polyunsaturated fatty acids, cholesterol, and glucose contribute to the disruption of the cellular redox homeostasis. Collectively or individually, these effects create pro-oxidant conditions in cells. Oxidative stress can induce neuronal damage, modulate intracellular signaling, and can ultimately lead to neuronal death by apoptosis or necrosis. Emerging evidence indicates that homocysteine (Hcy), a non-protein amino acid naturally present in the plasma, is implicated as a risk factor for numerous diseases. In particular, increased levels of circulating Hcy have been recognized as an independent risk factor for the development of vascular disease(s). Recent findings emphasize a relationship between elevated Hcy levels and neurodegeneration, which can be observed in Alzheimer's and Parkinson's diseases. An integrated response exists in the brain to detect and control diverse forms of stress. This is accomplished by a complex network of the so-called longevity assurance processes, which are controlled by several genes termed "vitagenes." Among these, the heat-shock proteins (HSPs) form a highly conserved system that is responsible for the preservation and repair of the correct protein conformation. Recent studies have shown that the heat-shock response (HSR) contributes to cytoprotection in a number of human diseases including inflammation, cancer, aging, and neurodegenerative disorders. Given the broad cytoprotective properties of the HSR, interest mounts currently among investigators toward discovering and developing pharmacological agents capable of inducing HSR. L: -Acetylcarnitine (LAC) is proposed as a therapeutic agent for several neurodegenerative disorders and also current evidence suggests that the compound may play a critical role in the modulation of cellular stress response in health and disease conditions. Here, we review the emerging salient concepts highlighting the pathways of neurodegeneration and the role of LAC in modulating the redox-dependent mechanisms responsible for the upregulation of vitagenes in brain that leads to the enhancement of stress tolerance in brain.

A 29 year old woman presented with an intracerebral hemorrhage. Angiographic findings were consistent with unilateral moyamoya. The patient was managed non-surgically and discharged with the indication of periodical followup angiography. Moyamoya is a rare entity that must be considered in the differential diagnosis of ischemic or hemorrhagic cerebrovascular events. At present, the natural history of unilateral moyamoya is not well established in relation to the progression to a bilateral form and to rebleeding risk. Periodical follow-up angiography (conventional or MRI) seems a reasonable management strategy.

During the development of the nervous system, a large number of neurons are eliminated through naturally occurring neuronal death. Many morphological and biochemical properties of such dying neurons are reminiscent not only of apoptosis, a type of death involving the action of genetically programmed events, but also of epigenetic phenomena such as oxidative stress. Increasing evidence demonstrates that oxidative stress alters the expression of antioxidant enzymes and enhances expression and/or DNA binding of numerous transcription factors, including heat shock factor. The latter is a transcription factor for specific promoter elements located upstream of the heat shock genes. Heat shock proteins (Hsps) are essential, highly conserved proteins that are needed for normal cell growth and maintenance, and expression of Hsps has been detected during embryogenesis in various organisms. Developmental profiles of Hsps indicate that they are differentially regulated during neural maturation, suggesting a role for Hsps in neural cell differentiation. Their putative function in cell remodeling during migration and differentiation, as well as during postnatal development, a time of extensive cell differentiation, is considered in the present review. Moreover, the function of Hsps in cell signaling, protein transport and the effect of heat shock on neural plate induction and brain development are discussed.

Recent evidence suggests a link between the aberrant re-expression of cell cycle proteins in adult neurons of the Alzheimer's disease brain and the process of apoptotic degeneration. Here we will discuss this unexpected phenomenon as related to the mechanisms of beta-amyloid toxicity, and its significance for therapeutic possibilities.

Nitric oxide (NO) has been implicated as a potential contributor to neural cell death in a variety of neurological conditions. Glial cells in the CNS can produce nitric oxide in response to cytokines, and it is known that neurons, which are characterized by low levels of natural antioxidants and free radical scavengers, are more susceptible to oxidative damage. Heme oxygenase-1 (HO-1) is a cellular stress protein (HSP32) which is expressed in several tissues upon stimulation by a large number of potentially noxious stimuli, including oxidative stress. Activation of HO-1, generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury.

Aggregates of beta-amyloid peptide (betaAP), the main constituent of amyloid plaques in Alzheimer's brain, kill neurons by a not yet defined mechanism, leading to apoptotic death. Here, we report that both full-length betaAP((1-40)) or ((1-42)) and its active fragment betaAP((25-35)) act as proliferative signals for differentiated cortical neurons, driving them into the cell cycle. The cycle followed some of the steps observed in proliferating cells, including induction of cyclin D1, phosphorylation of retinoblastoma, and induction of cyclin E and A, but did not progress beyond S phase. Inactivation of cyclin-dependent protein kinase-4 or -2 prevented both the entry into S phase and the development of apoptosis in betaAP((25-35))-treated neurons. We conclude that neurons must cross the G1/S transition before succumbing to betaAP signaling, and therefore multiple steps within this pathway may be targets for neuroprotective agents.-Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Giuffrida Stella, A. M., Canonico, P. L., Nicoletti, F., Sortino, M. A. Mitotic signaling by beta-amyloid causes neuronal death.

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPα, β, and γ, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1–14; Zelenika et al. [1995] Mol. Brain Res. 30:251–258), we called this novel mRNA isoform GFAPδ. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPδ transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (α, β, and γ) produce hypothetical translation products differing in the aminoterminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain. J. Neurosci. Res. 56:219–228, 1999. © 1999 Wiley-Liss, Inc.

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.

The level of the mRNA for glial fibrillary acidic protein (GFAP), the major protein of the intermediate filaments of astroglial cells, and the activity of glutamine synthetase (GS), an enzyme selectively localized in astrocytes, were measured at different times after a unilateral mechanical lesion in the rat cerebral cortex. A rapid and early increase (6 hours post-lesion) in GFAP mRNA was observed; GFAP mRNA level reached a peak at 1-3 days and then decreased. Moreover, an astrocytic activation in cortical zones far from the injury site and in the contralateral hemisphere was detected. No change of GS activity was observed in the same model of brain injury, showing that this astroglial marker is not modified during the reactive gliosis obtained with this experimental model. GFAP mRNA has also been detected in the rat sciatic nerve; however, its level was not modified after nerve transection, suggesting a different regulation of GFAP expression in the peripheral nervous system.

Oxidative damage is the major responsible for mitochondrial dysfunction during aging and many neurodegenerative diseases with respiratory chain Complex I mostly affected. A down-regulation of the mitochondrial respiratory chain Complex-I subunits ND1 (mitochondrial encoded) in rat total cerebral cortex RNA and 39 kDa (nuclear encoded) in frontal cortex protein extracts was observed at 6, 12 and 28 months of age. However the 39 kDa subunit showed an age-related up-regulation in the parietal cortex and no changes in the cerebellum protein extracts. In frontal and parietal cortex protein extracts a significant reduction of subunit α (nuclear encoded) of FoF1-ATP synthase, with no changes in the cerebellum, were also observed. A similar impairment was found also for mitochondrial adenine nucleotide translocator ANT-1 in total rat cerebral cortex RNA. Any change of subunit synthesis might lead to neuronal dysfunction and increase of ROS.

An increase in astrocyte mitogenic factors and in some specific astroglial enzymatic activities after neuronal injury has been observed. Our study is concerned with the effect of the intracerebral administration of ibotenic acid (IBO) into the rat hippocampus. IBO injection causes a selective degeneration of neurons while sparing afferent fibers. We observed a transient increase in glutamine synthetase activity, a well-known astroglial marker, reaching a peak at 9-15 days after injury in lesioned hippocampus. We investigated the presence of astrocyte mitogenic factors at various times after toxin injection. Crude extracts, prepared from lesioned hippocampi 4, 9, and 14 days after IBO injection, were tested for the ability to stimulate [methyl-3H]thymidine incorporation into rat astroglial cell cultures. Crude extracts prepared 9 and 14 days after IBO injection showed a higher mitogenic activity compared to extracts prepared 4 days after lesion. Mitogenic activity of injured brain extracts was suppressed by heat inactivation (100 degrees C for 10 min).

An analytical method for the determination of saturated very long chain (VLC) fatty acids in the serum has been devised. Free fatty acids obtained after hydrolysis of total lipid extracts were converted into p-bromophenacyl esters. The derivatives were purified in two sequential steps by clean-up on C18 reversed-phase cartridge and fractionation by reversed-phase thin-layer chromatography (TLC), and then quantitated by high performance liquid chromatography (HPLC) analysis. This technique provides a reliable and alternative method for the biochemical identification of patients and carriers of an inherited metabolic disease characterized by the accumulation of saturated VLC fatty acids (C24-C26) such as Adrenoleukodystrophy (ALD). In four cases of diagnosed ALD the fatty acid composition of serum total lipids was dramatically enriched in saturated VLC fatty acids compared to controls. The ratio of hexacosanoic acid (C26:0) to docosanoic acid (C22:0) in ALD patients was approximately six-fold higher than that of healthy controls or patients affected by metabolic or neurological disorders other than ALD.

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-14C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed. The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging. In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-3H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured. The results obtained showed that the incorporation of [3H]thymidine into DNA and of [14C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development; however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [3H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.

The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15–30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl-3H]thymidine or [5, 6 -3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37°C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl-3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr. The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4–10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied. Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures. The addition of EGF to DMEM + BSA significantly increased cell number, while addition of insulin had no effect. The highest values for cell number were observed when insulin and transferrin were added together with EGF. Moreover, EGF treatment in the presence of serum significantly increased the incorporation of labeled amino acids into vimentin, glial fibrillary acidic protein, and actin.

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.

SUMMARYThe ultrastructure of the whole cellular system which forms the absorbing trichome of Tillandsia usneoides L. was studied under conditions of water stress and moisture. Cytological modification which are associated with so special a modality of functioning, were identified. The trichome is tipically divided into two parts: the «shield» whose cells form a central disk and a wing; the «stalk» whose structure includes a dome cell, two intermediate cells and two foot cells. All the walls of the absorbing system cells did not show any differences under the two physiological conditions assayed. No differences were observed also between the cytoplasm of the «shield» cells in the two experimental conditions. However, the cytoplasm of the central disk cells remained vital implying that they play an active role under both conditions, in this portion of the trichome, as regards the choise of nutrient solutions. The most important ultrastructural aspects were offered by the cytoplasm of «stalk» cells under bot...

This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures. After 12 hours of EGF treatment in serum-free medium or chemically defined medium, significant increase in RNA labeling, and also in acid-soluble radioactivity and RNA content, was observed. The ratio RNA/DNA was significantly higher in EGF-treated cultures compared with controls. Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF-treated cultures. Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95-83 and 44 kd, which were significantly more labeled in EGF-treated cultures.

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [3H]uridine) and in vitro (either by incubation of tissue slices with [3H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg2+-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn2+-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl-CoA and of [gamma-32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low-molecular-weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000-90,000 daltons)--absent at 1 day, appearing at 10 days, and more evident at 30 days of age--was observed in both neuronal and glial nuclei.

An axolemma-enriched fraction was isolated and characterized from homogenates of brain stem, pooled optic nerve and tract, and sciatic and hypoglossal nerves of adult rabbits. In these fractions, the phospholipase A1 and A2, as well as the activity of acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine and acyl-CoA:2-acyl-sn-glycero-3-phosphorylcholine acetyl transferase, using 1-acyl- and 2-acyl-GPC as acyl acceptors, were studied. The activity of the four enzymes was clearly detectable in the central nervous system (CNS) and peripheral nervous system (PNS) axolemmatic preparations, as well as in other subcellular fractions examined. The axolemma fractions, in which acetylcholinesterase displayed the highest activities, were particularly enriched in the acylation reaction enzymes. These latter showed specific activities about twofold higher compared with those of the homogenates and significant correlation with acetylcholinesterase. The noticeable presence of these enzyme activities in both CNS and PNS axolemma suggests that a deacylation-reacylation system for phospholipids may be operative in this membrane.