Characterization of Cellulosic Pulps Isolated from Two Widespread Agricultural Wastes: Cotton and Sunflower Stalks.

Globally, huge amounts of cotton and sunflower stalks are generated annually. These wastes are being underutilized since they are mostly burned in the fields. So, in this work, we proposed a three-step method consisting of acid pre-treatment, alkaline hydrolysis, and bleaching for the extraction of cellulose pulps. These pulps were characterized to assess their morpho-structural and thermal properties. The design of experiments and response surface methodology were used for the optimization of the acid pre-treatment in order to achieve maximum removal of non-cellulosic compounds and obtain pulps enriched in cellulose. For cotton stalks, optimal conditions were identified as a reaction time of 190 min, a reaction temperature of 96.2 °C, and an acid (nitric acid) concentration of 6.3%. For sunflower stalks, the optimized time, temperature, and acid concentration were 130 min, 73.8 °C, and 8.7%, respectively. The pulps obtained after bleaching contained more than 90% cellulose. However, special care must be taken during the process, especially in the acid pre-treatment, as it causes the solubilization of a great amount of material. The characterization revealed that the extraction process led to cellulose pulps with around 69-70% crystallinity and thermal stability in the range of 340-350 °C, ready to be used for their conversion into derivatives for industrial applications.

Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Obtaining Cellulose Nanocrystals from Olive Tree Pruning Waste and Evaluation of Their Influence as a Reinforcement on Biocomposites.

The objective of this work is to improve the mechanical properties of polylactic acid (PLA) by incorporating cellulose nanocrystals (CNC) previously obtained from a cellulose pulp extracted from olive tree pruning (OTP) waste. Composites were manufactured by melt processing and injection moulding to evaluate the effect of the introduction of CNC with conventional manufacturing methods. This OTP-cellulose pulp was subjected to a further purification process by bleaching, thus bringing the cellulose content up to 86.1%wt. This highly purified cellulose was hydrolysed with sulfuric acid to obtain CNCs with an average length of 267 nm and a degradation temperature of 300 °C. The CNCs obtained were used in different percentages (1, 3, and 5%wt.) as reinforcement in the manufacture of PLA-based composites. The effect of incorporating CNC into PLA matrix on the mechanical, water absorption, thermal, structural, and morphological properties was studied. Maximum tensile stress and Young's modulus improved by 87 and 58%, respectively, by incorporating 3 and 5%wt. CNC. Charpy impact strength increased by 21% with 3%wt. These results were attributed to the good dispersion of CNCs in the matrix, which was corroborated by SEM images. Crystallinity index, glass transition, and melting temperatures were maintained.

Estrogens influence differentially on the pelvic floor muscles activation at somatovisceral reflexes involved in micturition of rabbits.

OBJECTIVE: To determine the estrogen-dependency of the bladder and urethral function and the coordinated activation of pelvic floor muscles (PFM) during micturition. METHODS: We allocated age-matched female rabbits to control, 1-month ovariectomized (OVX), and OVX plus 2-week estradiol benzoate (EB) groups to record cystometry, urethral pressure, and electromyograms of bulbospongiosus (Bsm), and pubococcygeus muscles (Pcm) simultaneously. We also measured serum estradiol levels and myofiber cross-sectional area. We assessed urodynamic and urethral variables, categorized the Bsm-Pcm activation patterns at storage and voiding phases, and obtained the power spectrum density of muscle activation around the voiding phase. We investigated the influence of ovarian hormones, in general, and the contribution of estrogen, particularly on the functions of the bladder, urethra, and PFM. Statistical significance was set at P < 0.05. RESULTS: Ovarian hormones influence the bladder, urethral, and PFM functions. The urodynamics analyses indicated estrogens contribute to voiding duration and, to a lesser extent, to the time between bladder contractions. Urethral pressure at closure (maximal pressure-to-maximal urethral pressure ratio) improved partially (8%, P < 0.05) in the OVX plus 2-week estradiol benzoate compared with OVX, but urethral resistance increased (∼1.9-fold, P < 0.05) compared with control rabbits. Our findings support that Pcm activity at voiding is estrogen-sensitive, albeit EB administration reduced it at storage resume, which relates to high urethral resistance. CONCLUSIONS: Ovariectomy impairs bladder and urethral pressures and Bsm and Pcm activation at micturition in anesthetized rabbits. Estrogen administration partially reverts some of these effects and influences Pcm activation.

Differential estrogen-related responses in myofiber cross-sectional area of pelvic floor muscles in female rats.

OBJECTIVE: To determine the role of estrogens in myofiber cross-sectional area (CSA) of the pubococcyegeus (Pcm) and iliococcygeus muscles (Icm). METHODS: In Experiment 1, we excised the Pcm and Icm during the metestrus and proestrus stages of the estrous cycle to measure the myofiber CSA. In Experiment 2, we allocated other rats into the following groups: sham (Sh), ovariectomized (OVX), OVX plus 1,4,6-androstatriene-3,17-dione (ATD; OVX + ATD), an aromatase inhibitor, and OVX plus estradiol benzoate (OVX + EB). We carried out appropriate statistical tests to determine significant differences (p ≤ 0.05) in variables measured for both Experiments. RESULTS: The Pcm myofiber CSA at proestrus was higher than at metestrus, while the Icm myofiber CSA did not change. Ovariectomy increased the Pcm myofiber CSA, which was exacerbated with the ATD administration. The EB supplementation successfully reversed the ovariectomy-induced enlargement of the CSA. No significant changes were detected for the Icm myofiber CSA. CONCLUSIONS: Fluctuating ovarian steroid levels at the estrus cycle significantly influence the CSA myofiber of the Pcm but not that of the Icm. Estrogen actions, having a gonadal or extragonadal origin, influence importantly the CSA of the Pcm.

Lysine Restriction and Pyridoxal Phosphate Administration in a NADK2 Patient.

We report the case of a 10-year-old Spanish girl with mutations in NADK2 Prenatal central nervous system abnormalities showed ventriculomegaly, colpocephaly, and hypoplasia of the corpus callosum. At birth, axial hypotonia, uncoordinated movements, microcephaly, and generalized cerebellar atrophy were detected. Metabolic investigations revealed high lysine, lactate, and pipecolic acid levels in blood and cerebrospinal fluid. Pyruvate carboxylase and pyruvate dehydrogenase activity in fibroblasts were normal. Beginning at birth she received biotin, thiamine, and carnitine supplementation. A lysine-restricted diet was started when she was 1 month old. Because pipecolic acid was high, pyridoxine was added to treatment. At 3 years old, astatic myoclonic epilepsy appeared, with no response to levetiracetam. We switched pyridoxine to pyridoxal phosphate, with electroclinical improvement. Because the activity of mitochondrial respiratory chain complexes III and IV was slightly low in muscle, other cofactors such as ubidecarenone, idebenone, vitamin E, and creatine were added to the treatment. At 8 years old, plasma acylcarnitine testing was performed, and high levels of 2-trans, 4-cis-decadienoylcarnitine were found. Whole exome sequencing identified a homozygous splice site mutation in NADK2 (c.956+6T>C; p.Trp319Cysfs*21). This substitution generates exon skipping, leading to a truncated protein. In fact, NADK2 messenger RNA and the corresponding protein were almost absent. Now, at 10 years of age she presents with ataxia and incoordination. She has oromotor dysphasia but is able to understand fluid language and is a very friendly girl. We hypothesize that the patient's clinical improvement could be due to her lysine-restricted diet together with cofactors and pyridoxal phosphate administration.

Pyridoxal 5'-phosphate values in cerebrospinal fluid: reference values and diagnosis of PNPO deficiency in paediatric patients.

Our aim was to establish reference values for cerebrospinal fluid (CSF) pyridoxal 5'-phosphate (PLP) in a paediatric population for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency. For reference values, CSF samples from 113 paediatric controls (age range: 1 day-18 years) from Barcelona and London were analysed. Cerebrospinal fluid PLP and biogenic amine concentrations were analysed by HPLC with fluorescence and electrochemical detection. Pyridoxal 5'-phosphate concentrations in 4 patients with PNPO deficiency were determined. A negative correlation between CSF PLP values and age of controls was observed in both populations (r=-0.503; p<0.0001 and r=-0.542; p=0.002). Reference values were stratified into 4 (Barcelona) and 3 age groups (London). For the newborn period, CSF PLP reference intervals were 32-78 and 44-89 nmol/L for the Barcelona and London centers, respectively). No correlation was observed in the different age groups between PLP values and biogenic amines metabolites. PLP values in neonates with PNPO deficiency were clearly decreased (PLP=3.6, 12.0, 14.0 and 18.0 nmol/L) compared with our reference ranges. In conclusion, reference values for CSF PLP should be stratified according to age. No association was observed between PLP values and biogenic amines metabolites. In our 4 cases with PNPO deficiency, CSF PLP values were clearly below the reference values.

A new fatal case of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency.

We present a patient with severe pyridox(am)ine 5'-phosphate oxidase deficiency and homozygosity for a novel nonsense-mutation, p.A174X, in the PNPO gene who died with pyridoxal phosphate (PLP) treatment despite initial clinical recovery. He presented neonatally, with the classical clinical symptoms of the disease. Increase of urinary vanillactate was the first biochemical factor of alert. Amino acid and neurotransmitter analysis in CSF indicated reduced activity of several PLP-dependent enzymes. The diagnosis was confirmed by mutational studies. From this and the other reported patients it may be concluded that the administration of PLP should not be delayed until the complete biochemical evidence is obtained.

[Usefulness of analysis of cerebrospinal fluid for the diagnosis of neurotransmitters and pterin defects and glucose and folate transport deficiencies across blood brain barrier]. / Utilidad del análisis del líquido cefalorraquídeo para el estudio de las deficiencias del metabolismo de neurotransmisores y pterinas y del transporte de glucosa y folato a través de la barrera hematoencefálica.

BACKGROUND AND OBJECTIVE: In the last few years, it has been described inborn errors of neurotransmitter and pterin metabolism and defects in folate and glucose transport across blood brain barrier. All these defects are classified as rare diseases and needs cerebrospinal fluid (CSF) sample analysis for diagnosis. Our aim was to evaluate the results of the application of a CSF analysis protocol in a pediatric population from Spain and Portugal presenting with neurological disorders of unknown origin. PATIENTS AND METHOD: We studied CSF samples from and 283 patients with neurological disorders of unknown origin and 127 controls. Neurotransmitters were analysed by HPLC with electrochemical detection, and pterins and 5-methyltetrahydrofolate were determined by HPLC with fluorescence detection. RESULTS: We diagnosed 3 patients with tyrosine hidroxylase deficiency, 2 with dopa responsive dystonia, 14 with GTP-ciclohydrolase deficiency, 2 with glucose transport deficiency and 43 with cerebral folate deficiency. CONCLUSIONS: This study allowed us to diagnose new patients, and more importantly, the establishment in all of them of a pharmacological or nutritional treatment. The most frequent defect found was CSF 5-methyltetrahydrofolate deficiency, which was present in different groups of patients.

Utilidad del análisis del líquido cefalorraquídeo para el estudio de las deficiencias del metabolismo de neurotransmisores y pterinas y del transporte de glucosa y folato a través de la barrera hematoencefálica / Usefulness of analysis of cerebrospinal fluid for the diagnosis of neurotransmitters and pterin defects and glucose and folate transport deficiencies across blood brain barrier

Fundamento y objetivo: En la última década se ha descrito diferentes errores congénitos del metabolismo de los neurotransmisores (NT), en especial de las vías dopaminérgica y serotoninérgica y de las pterinas. También se ha descrito defectos primarios en el transporte de glucosa y 5-metiltetrahidrofolato (5-MTHF) a través de la barrera hematoencefálica, todos ellos enfermedades raras para cuyo diagnóstico es necesario el estudio en líquido cefalorraquídeo (LCR). Nuestro objetivo ha sido evaluar los resultados de la aplicación de un protocolo de análisis de LCR en España y Portugal durante 3 años en pacientes pediátricos con trastornos neurológicos de origen desconocido. Pacientes y método: Se estudió a 127 individuos control y 283 pacientes con trastornos neurológicos de origen desconocido. El análisis de NT se realizó mediante HPLC con detección electroquímica y el análisis de pterinas y 5-MTHF, mediante HPLC con detección de fluorescencia. Resultados: Se ha diagnosticado 3 deficiencias de tirosina hidroxilasa en una misma familia, 2 casos de distonía sensible a L-dopa, 2 familias con defiencia de guanosinatrifosfato-ciclohidrolasa dominante (14 casos), 2 deficiencias del transportador de glucosa y 43 deficiencias de folato en LCR. Conclusiones: Este estudio ha permitido el diagnóstico de nuevos pacientes y, lo que es más importante, el establecimiento en todos ellos de un tratamiento farmacológico o nutricional. Las deficiencias de 5-MTHF han sido las más frecuentes y se las ha detectado en diferentes grupos de pacientes

Background and objective: In the last few years, it has been described inborn errors of neurotransmitter and pterin metabolism and defects in folate and glucose transport across blood brain barrier. All these defects are classified as rare diseases and needs cerebrospinal fluid (CSF) sample analysis for diagnosis. Our aim was to evaluate the results of the application of a CSF analysis protocol in a pediatric population from Spain and Portugal presenting with neurological disorders of unknown origin. Patients and method: We studied CSF samples from and 283 patients with neurological disorders of unknown origin and 127 controls. Neurotransmitters were analysed by HPLC with electrochemical detection, and pterins and 5-methyltetrahydrofolate were determined by HPLC with fluorescence detection. Results: We diagnosed 3 patients with tyrosine hidroxylase deficiency, 2 with dopa responsive dystonia, 14 with GTP-ciclohydrolase deficiency, 2 with glucose transport deficiency and 43 with cerebral folate deficiency. Conclusions: This study allowed us to diagnose new patients, and more importantly, the establishment in all of them of a pharmacological or nutritional treatment. The most frequent defect found was CSF 5-methyltetrahydrofolate deficiency, which was present in different groups of patients

Acute and chronic hypoglycemic effect of Ibervillea sonorae root extracts-II.

Ibervillea sonorae's root, or "wareque" (Cucurbitaceae), is widely used in Mexican traditional medicine for the control of diabetes mellitus. In the present study, the hypoglycemic effects produced by the acute and chronic administration of various extracts of Ibervillea sonorae were investigated. Both the traditional preparation (aqueous decoction) and the raw extract (juice) from the root resulted in significant reductions of glycemia in healthy mice after intraperitoneal administration at a dose of 600 mg/kg. Additionally, ground dried root was used to obtain a dichloromethane (DCM) extract and a methanol (MeOH) extract. The DCM extract induced a clear reduction of glycemia in healthy (P < 0.05) and in alloxan-diabetic mice. The intraperitoneally administered DCM extract caused a severe hypoglycemia that produced lethality in all the treated animals when doses of 300 and 600 mg/kg body weight were used. Since the DCM extract showed a marked hypoglycemic activity, it was administered daily per os to alloxan diabetic rats, employing corn oil and tolbutamide as controls. After 41 days of DCM extract administration at a dose of 300 mg/kg/day, diabetic rats showed improvement in glycemia, body weight, triglycerides, and GPT in comparison with the diabetic control group. Total cholesterol, GOT, and uric acid blood levels were not affected.

Accuracy of noninvasive estimates of respiratory muscle effort during spontaneous breathing in restrictive diseases.

Mean inspiratory pressure (Pi), estimated from the occlusion pressure at the mouth and the inspiratory time, is useful as a noninvasive estimate of respiratory muscle effort during spontaneous breathing in normal subjects and patients with chronic obstructive pulmonary disease. The aim of this study was to compare the Pi with respect to mean esophageal pressure (Pes) in patients with restrictive disorders. Eleven healthy volunteers, 12 patients with chest wall disease, 14 patients with usual interstitial pneumonia, and 17 patients with neuromuscular diseases were studied. Pi, Pes, and mean transdiaphragmatic pressure were simultaneously measured. Tension-time indexes of diaphragm (TTdi) and inspiratory muscles (TTmu) were also determined. In neuromuscular patients, significant correlations were found between Pi and Pes, Pi and transdiaphragmatic pressure, and TTmu and TTdi. A moderate agreement between Pi and Pes and between TTmu and TTdi was found. No significant correlation between these parameters was found in the other patient groups. These findings suggest that Pi is a good surrogate for the invasive measurement of respiratory muscle effort during spontaneous breathing in neuromuscular patients.

Pure partial trisomy 7q: two new patients and review.

We report on two new cases with a pure partial trisomy of the long arm of chromosome 7. Patient 1 was a female who showed cleft palate with retrognathia, cardiomyopathy, and pulmonary hypertension. Patient 2 was a male who showed microretrognathia, cleft palate, micropenis, camptodactyly, and clynodactyly. High-resolution G-bands (550-850) karyotype showed that patient 1 had an extra chromosome, which resulted from the adjacent 3:1 segregation from a maternal balanced reciprocal translocation, and patient 2 had an abnormaly Y chromosome. Fluorescent in Situ Hybridization (FISH) analysis with a whole chromosome painting confirmed in the first patient that the extra chromosome was from chromosome 7, and in patient 2 the abnormal Y chromosome had extra material of chromosome 7 origin. Three different clinical entities have been described as the product of the partial trisomy of three different 7q regions, although some authors have found no karyotype-phenotype correlations. Of the patients presented here, patient 1 had trisomy of those three regions, and patient 2 had trisomy of two of those regions.