Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide.

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (∼100nm) and positively charged (+28.6mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000™, but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.

Characterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector.

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid Lipofectamine™. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa.

The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes.

Overexpression and purification of PWL2D, a mutant of the effector protein PWL2 from Magnaporthe grisea.

The rice blast disease caused by the ascomycete Magnaporthe grisea continues to cause a tremendous impact in rice (Oryza sativa) cultures around the world. Elucidating the molecular basis of the fungus interactions with its host might help increase the general understanding of the pathogen-host relationship. At the moment of invasion, the fungus secretes effectors that modify host defenses and cellular processes as they successively invade living rice cells. PWL2, an effector protein, is a known AVR (avirulence) gene product. The PWL2 gene prevents the fungus from infecting weeping lovegrass (Eragrostis curvula). In this study, we identified a PWL2 allele gene (which we termed PWL2D) in a strain of M. grisea. The sequence of PWL2D has only two bases different from that of PWL2, producing alterations in residue 90 and residue 142. However, the alteration of residue 90 (from D(90) to N(90)) is critical to gene function. Here, we cloned the gene PWL2D in a pET System vector, expressed the gene product in Escherichia coli and evaluated by spectroscopic techniques some aspects of the PWL2D structure. While TRX-tagged PWL2D is prone to aggregation, the solubility of PWL2D is improved when it is overexpressed without its original signal peptide. Expression and purification procedures for these constructs are described. Finally, we found out that the protein seems to be an intrinsically disordered protein. Results from these studies will facilitate structural analysis of PWL2D and might contribute to understanding the gene's function and of fungal/plant interactions.

On the stability of plasmid DNA vectors during cell culture and purification.

Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4 degrees C, >12 weeks at -20 degrees C) prior to processing. With alkaline lysates, however, storage at -20 degrees C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization.

Movement and mood disorder in two brothers with Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder with a wide spectrum of phenotypic presentations. We report the case histories of two adult brothers with GD who developed both parkinsonism and psychiatric symptoms. Direct sequencing and real-time polymerase chain reaction were used to establish that the patients were homozygous for mutation L444P. While parkinsonism has been described previously in GD, these patients had atypical features, including a complicated mood disorder. The comorbidity of GD and a mood disorder is a new finding, as psychiatric manifestations of GD have been described rarely. The etiology of the mental illness could be related to the processes contributing to the development of parkinsonism.

Tardive priapism associated with clozapine. A case report.

A man affected by schizoaffective disorder and alcohol abuse presented priapism after eleven years of clozapine treatment. After surgical intervention and resolution of priapism, he continued clozapine treatment at the same dose without problems. Clozapine withdrawal is not mandatory in patients who develop priapism in the course of treatment.

[A case of pervasive developmental disorder with chromosomal translocation (X; 4) (p11; q13)]. / Un cas de trouble envahissant du développement avec translocation chromosomique (X; 4) (p11; q13).

INTRODUCTION: Chromosomal aberrations, with or without congenital physical abnormalities, have been frequently found associated with neuropsychiatric disorders, including mental retardation, psychosis, autism, and criminal behaviour. The meaning of the association frequently remains unclear. However, consistent findings of association between specific chromosomal abnormalities and clinical phenotype may provide evidence of a causal relationship and shed light on the pathogenesis of obscure disorders. CASE-REPORT: Here, we present the case of a 28 year-old, Caucasian male affected by pervasive developmental disorder, associated with chromosomal translocation 46, XY, t (X; 4) (p11; q13), and abnormal facial features. A few days after birth, the patient was taken away from his parents and adopted for unknown reasons. No information is available about his biological relatives. Mild delay in the development of spoken language was reported. Since early childhood, the patient's behaviour was characterized by troublesome relationship with his parents and his fellows, and persistent violation of norms and rules at home and at school. Consequently, social and school functioning was poor. When he was eight, verbal and motor stereotypy appeared for the first time. As an adolescent, he was more and more aggressive. He exhibited countless episodes of rage and verbal and physical aggressiveness. After he had completed secondary school, his way of life was chaotic. He got into the habit of staying away from home, sleeping in the day and vagabonding at night. He began to abuse alcohol. Grandiosity and persecutory delusions became evident. He claimed to hate the Vatican, the Pope, and the Polish people and to be the Devil, the Antichrist. He feared that his food was poisoned by his mother and refused to eat at home any more. He loved to remain in a cage with two wild dogs, accumulating and keeping bottles full of his urine. He often engaged in violent fights in the street with tramps and foreigners. Finally, he was involuntary admitted to a psychiatric intensive care unit. He was hostile, uncooperative, and violent. Magnetic resonance imaging of brain was normal, Wechsler Adult Intelligence Scale IQ score was 96 (total), 108 (verbal), 80 (non verbal), and Standard Progressive Matrices score was 44/60, chromosomal examination [banding R (RBG)] revealed an apparently balanced translocation 46, XY, t (X; 4) (p11; q13). The patient was treated with risperidone (8 mg/day) and valproate (1500-2000 mg/day) with improvement. Psychotic symptoms, hostility and violence vanished. Amazingly, his behaviour and attitude became normal. Very early onset of symptoms, absence of negative signs, and dysmorphic features suggesting an underlying medical disease do not support the diagnosis of schizophrenia. DISCUSSION: The diagnosis of pervasive developmental disorder, not otherwise specified, could be made, considering the delay in the development of spoken language, the large discordance between verbal and non verbal WAIS IQ score, the presence of stereotypy, abnormal facial features, and motor clumsiness. The late onset of symptoms precludes the diagnosis of autism, while the delay in language does not permit the diagnosis of Asperger's disorder. The lack of information on his biological relatives did not permit us to assess the presence of genetic, physical or mental abnormalities in his family. Therefore, the causal relationship between the chromosomal translocation and the psychiatric disorder is uncertain in this patient. Similar genetic abnormalities found in patients affected by neuropsychiatric disorders could confirm an etiological link.

Asperger's disorder in the emergency psychiatric setting.

Asperger's syndrome (AS) is a pervasive developmental disorder that may be unrecognized, especially if signs of other psychiatric disorders coexist. The objectives of this paper are: 1) to ascertain the prevalence of AS in the emergency psychiatric setting; and 2) to describe features of AS which may help to differentiate these patients from patients with psychotic disorders. Among 2500 patients admitted to a psychiatric intensive care unit, 5 (0.2%) received a diagnosis of AS, for the first time. Besides impairment of social interaction, common features were the following: male gender, left handedness, obsessive-compulsive symptoms, cognitive hyper-abilities, violent behavior, sense of humor, low WAIS total score, high WAIS verbal/performance score ratio, unusual, restricted interest and clumsiness. Comorbid schizophrenia is difficult to rule out in these patients. Psychotic symptoms should not be overvalued in making the diagnosis when specific features of AS are present.

Neurocutaneous melanosis and psychosis: a case report.

The paper describes a case of neurocutaneous melanosis (NM), with mental retardation, chronic psychosis, and epilepsy possibly due to a temporal focus. This is the first report of NM associated with a severe and chronic psychosis. It is likely that such an association has not previously been described because of the ominous prognosis of most cases of NM with early involvement of the central nervous system.

Novel antipsychotics and acute dystonic reactions.

The growing use of atypical antipsychotics has led to a decrease of acute dystonic reactions (ADR). To evaluate the prevalence of ADR, we recorded all ADR occurring in a population of patients consecutively admitted to a psychiatric intensive care unit. Among 1337 cases treated with antipsychotics, we observed 41 cases (3.1%) affected by ADR. At discharge, mean chlorpromazine-equivalent daily dose was 465.8 (+/-421.5) mg, while 39 cases (3.0%), all treated with typical neuroleptics, received anticholinergics. During hospitalization, 15 cases received quetiapine, 19 sertindole, 95 olanzapine, 142 clozapine, 495 risperidone and 561 typical neuroleptics. Four ADR occurred among the cases treated with risperidone monotherapy, and 4 occurred in risperidone-treated patients after emergency parenteral treatment with typical neuroleptics. In these last 4 cases, temporal relationship suggested that typical neuroleptics had caused ADR. One ADR occurred in a patient treated with olanzapine and 1 ADR in a patient treated with quetiapine. Among cases assuming typical neuroleptics, 32 ADR occurred. The difference between typical and atypical neuroleptics is highly significant (chi2 = 27.756; d.f. = 1; p = 0.000). Atypical antipsychotics carry a minimal risk of ADR.

Second-generation antipsychotics in the emergency care setting. A prospective naturalistic study.

The objective of this subject was to examine the impact of the replacement of standard neuroleptics with atypical antipsychotic agents in an intensive psychiatric care unit. A mirror-image study was conducted. Cases admitted in the first semester of the year (when most of patients were treated with standard neuroleptics) were compared to cases admitted in the second semester of the year, when atypical antipsychotic agents were routinely utilized as first line treatment of patients with psychotic signs. Cases admitted in the first semester received a significantly higher daily dosage of antipsychotic drugs and more frequently received anticholinergics. In the second semester, a significantly higher number of patients received anticonvulsants, in particular valproate and gabapentin. There was no significant difference between the two groups of cases in the number of patients treated with antipsychotics, benzodiazepines, lithium, and carbamazepine and in the mean daily dose of benzodiazepines, lithium, carbamazepine, or valproate on the first day of hospitalization, the day of evaluation, and on discharge. On discharge, similar percentages of patients went home, were transferred to other Psychiatric Intensive Care Units (PICUs) or to private clinics, or left our PICU against medical advice. The length of hospitalization was similar in the two groups. There was no significant difference in the rate of aggressive or violent behavior registered in the two groups of cases. The risk of increasing violence rates, lengthening hospitalization, and facilitating patients' non-compliance should not be major concerns for physicians prescribing second-generation antipsychotics in the emergency care setting. Since these drugs have been shown to have at least similar efficacy (or greater efficacy in the case of clozapine) in the treatment of psychotic disorders as typical neuroleptics and to have a better side-effects profile, they should become first line treatment for patients with psychotic signs admitted to emergency care psychiatric facilities.

Recovery of cellulase by HPMC-salt precipitation: analysis by statistical experimental design.

Production of industrial enzymes including cellulases requires minimum cost with the downstream processing. The objective of this work was to analyze the precipitation of cellulases by ammonium sulfate in the presence of hydroxypropyl(methylcellulose) as a co-precipitant through the use of statistical experimental design. The model generated with the experimental results showed that high protein recovery can be achieved at high levels of temperature, aging times, and rate of salt-solution addition, and at a low mixing level. The results also allowed the observation that activity recovery was improved at high levels of temperature, rate of salt addition and mixing level, and a low level of aging time.

Frequency, phenomenology and anatomical-clinical correlates of major post-stroke depression.

BACKGROUND: The meaning of post-stroke depression is controversial. AIMS: To investigate the hypothesis that major post-stroke depression (PSD) may be due to organic factors (left frontal lesions) immediately after the stroke, but to psychosocial factors in later stages. METHOD: We studied 153 consecutive stroke patients, categorised on the basis of time elapsed since stroke, lesion location and presence/absence of major PSD. Fifty-eight were examined in the first two months following the stroke, 52 between two and four months, and 43 after four months or more. The symptom profiles and anatomical-clinical correlates of major PSD were studied in each subgroup. A group of 30 patients affected by a functional form of major depression were also investigated. RESULTS: The symptom profiles and anatomical-clinical correlates of major PSD were not different in the acute and more chronic stages. Clear symptom differences were, however, observed between major PSD and endogenous major depression. Motivated (reactive) symptoms prevailed in the former, whereas unmotivated symptoms prevailed in the latter. CONCLUSIONS: Our data are more consistent with a psychological than with a neurological model of post-stroke depression.

Diabetes insipidus and polydipsia in a patient with Asperger's disorder and an empty sella: a case report.

The paper describes a patient with Asperger disorder, Neurogenic Diabetes Insipidus (NDI) and Primary Empty Sella (ES). His response to vasopressin treatment suggested a concomitant presence of primary polydipsia. This is the first reported case of an autistic spectrum disorder associated with NDI or ES. The implications of the observed co-occurrence of these relatively rare disorders are discussed in relation to diagnosis and pathogenesis.

Relation of lesion location to verbal and nonverbal mood measures in stroke patients.

BACKGROUND AND PURPOSE: The aim of the present study was to evaluate the relation between poststroke depression and lesion location, avoiding previous methodological shortcomings. In particular, we intended to determine whether patients with left frontal lesions showed the highest depression scores. METHODS: Patients in the study, categorized on the basis of lesion location, included 149 stroke patients with lesions located in the anterior, central, or posterior regions of the right or left hemisphere. Verbal and nonverbal mood measures as well as the Hamilton Depression Scale Overall Score were the dependent measures of our investigation. Furthermore, the number of patients who could not be assessed or could be evaluated only with the nonverbal mood measure due to the presence of severe language disorders was recorded. RESULTS: No significant relation was observed between depressed mood and lesion location. Approximately one quarter of the left brain-damaged patients were partially or totally excluded from the study because of severe language disorders. CONCLUSIONS: Our data appeared to show that when methodological pitfalls and selection bias are carefully controlled, left frontal lesions are not a major determinant of poststroke depression.

Sexual behavior and hemispheric laterality of the focus in patients with temporal lobe epilepsy.

Various studies have described an unusually common incidence of sexual and reproductive dysfunction in patients affected by temporal lobe epilepsy (TLE). The purpose of the present study was to further investigate, by means of an ad hoc questionnaire, the relationship between sexual disorders and the hemispheric laterality of the epileptic focus in men and women with right (R) TLE and left (L) TLE. The results suggest a reduction of sexual interest in patients with R-TLE as compared with L-TLE in both men and women. This effect was fundamentally observed when sexual interest was implicitly explored. No significant difference was found between R-TLE and L-TLE groups concerning most aspects of sexual performance. Various hypotheses are discussed to interpret this effect of hemispheric lateralization on sexual interest.

The Post-Stroke Depression Rating Scale: a test specifically devised to investigate affective disorders of stroke patients.

Owing to the lack of instruments specifically constructed to study emotional and affective disorders of stroke patients, the nature of post-stroke depression (PSD) remains controversial. With this in mind, the authors constructed a new scale, the Post-Stroke Depression Scale (PSDS) which takes into account a series of symptoms and problems commonly observed in depressed stroke patients. The PSDS and the Hamilton Depression Rating Scale (HDS) were administered to a group of 124 patients, who had been classified, on the basis of DSM III-R diagnostic criteria, in the following categories: No depression (n = 32); Minor PSD (n = 47); Major PSD (n = 45). Scores obtained by these stroke patients on the PSDS and on the HDS were compared to those obtained on the same scales by 17 psychiatric patients also classified as major depression on the basis of DSM III-R diagnostic criteria. An analysis of the symptomatological profiles clearly showed that: (1) a continuum exists between the so-called "major" and "minor" forms of PSD; (2) in both groups of depressed stroke patients the depressive symptomatology seems due to the psychological reaction to the devastating consequences of stroke, since the motivated aspects of depression prevailed in depressed stroke patients, whereas the (biologically determined) unmotivated aspects prevailed in patients with a functional form of major depression; and (3) in stroke patients a DSM III-based diagnosis of major PSD could be in part inflated by symptoms (such as apathy and vegetative disorders) that are typical of major depression in a patient free from brain damage, but that could be due to the brain lesion per se in a stroke patient.