Stomatal response to leaf water potential in almond trees under drip irrigated and non irrigated conditions

Almond plants (Amygdalus communis L. cv. Garrigues) were grown in the field under drip irrigated and non irrigated conditions. Leaf water potential () and leaf conductance (g₁) were determined at three different times of the growing season (spring, summer and autumn). The relationships between and g₁ in both treatments showed a continuous decrease of g₁ as decreased in spring and summer. Data from the autumn presented a threshold value of (approx. –2.7 MPa in dry treatment, and approx. –1.4 MPa in wet treatment) below which leaf conductance remained constant.     

A relationship between the inhibition of heparan sulfate and chondroitin sulfate synthesis and the inhibition of molting by selenate in the hemipteran Rhodnius prolixus

The insect Rhodnius prolixus synthesizes heparan sulfate and chondroitin sulfate after a blood meal containing [35S]-inorganic sulfate. A 40 to 80% inhibition of heparan sulfate synthesis was obtained when the meal was supplemented with 10(-5) and 10(-4) M sodium selenate respectively. Likewise an inhibition of the molting in the order of 30 to 60% was observed when the insects were fed with blood containing 10(-5) and 10(-4) M selenate respectively. The insects after a subsequent meal without selenate molted normally. Except for the inhibition of the ecdysis no gross physiological or morphological changes could be observed in the insects. Based on these and other findings the possible role of sulfated glycosaminoglycans in the control of cell growth is discussed.     

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase

Background

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P₁ receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.

Methodology/Principal Findings

Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P_int, and attenuated S1P_ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P_int and potentiated motility of HPAECs to S1P_ext or serum. S1P_ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P_ext or 4-deoxypyridoxine-dependent endothelial cell motility.

Conclusions/Significance

These results suggest S1P_ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.     

Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene

The first mutant of Streptococcus pneumoniae showing a complete deletion in the lytA gene coding for the N-acetylmuramyl-L-alanine amidase has been isolated and characterized. This amidase was previously the only autolysin detected in this species. This mutant shows a normal growth rate and can be transformed using either chromosomal or plasmid DNA. The most remarkable biological consequences of the absence of the amidase are the formation of small chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In addition, this mutant exhibits a tolerant response against the beta-lactam antibiotics.     

Repetitive peptide boosting progressively enhances functional memory CTLs

Cytotoxic T lymphocytes (CTLs) play a critical role in controlling intracellular pathogens and cancer cells, and induction of memory CTLs holds promise for developing effective vaccines against critical virus infections. However, generating memory CTLs remains a major challenge for conventional vector-based, prime-boost vaccinations. Thus, it is imperative that we explore nonconventional alternatives, such as boosting without vectors. We show here that repetitive intravenous boosting with peptide and adjuvant generates memory CD8 T cells of sufficient quality and quantity to protect against infection in mice. The resulting memory CTLs possess a unique and long-lasting effector memory phenotype, characterized by decreased interferon-γ but increased granzyme B production. These results are observed in both transgenic and endogenous models. Overall, our findings have important implications for future vaccine development, as they suggest that intravenous peptide boosting with adjuvant following priming can induce long-term functional memory CTLs.     

Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.     

Neurocysticercosis: HP10 Antigen Detection Is Useful for the Follow-up of the Severe Patients

Background

The most severe clinical form of neurocysticercosis (NC) occurs when cysticerci are located in the subarachnoid space at the base of the brain (SaB). The diagnosis, monitoring and treatment of NC-SaB, constitutes a severe clinical challenge. Herein we evaluate the potential of the HP10 antigen detection enzyme-linked immunosorbent assay (HP10 Ag-ELISA) in the long term follow-up of NC-SaB cases. Assay performance was compared with that of Magnetic Resonance Imaging (MRI). In addition, the robustness of the HP10 Ag-ELISA was evaluated independently at two different institutions.

Methodology/Principal Findings

A double-blind prospective cohort trial was conducted involving 38 NC-SaB cases and a total of 108 paired serum and cerebrospinal fluid (CSF) samples taken at intervals of 4 to 8 months for up to 43 months. At each medical visit, results of sera and CSF HP10 Ag-ELISA and MRI obtained at last visit were compared and their accuracy was evaluated retrospectively, considering radiological evolution between appointments. In the long-term follow-up study, HP10 Ag-ELISA had a better agreement than MRI with retrospective radiological evaluation. High reproducibility of HP10 Ag-ELISA between laboratories was also demonstrated.

Conclusions

Results reported in this study establish for the first time the usefulness of the comparatively low cost HP10 Ag-ELISA for long term follow-up of NC-SaB patients.     

Congenital syphilis in Argentina: Experience in a pediatric hospital

In spite of being preventable, Congenital syphilis (CS) is still an important, and growing health problem worldwide. Fetal infection can be particularly aggressive, but newborns can be asymptomatic at birth and, if left untreated, develop systemic compromise afterwards with poor prognosis. We analyzed 61 CS diagnosis cases between 1987–2019 presenting at the Buenos Aires Children’ Hospital. The distribution of cases showed a bimodal curve, with a peak in 1992–1993 and in 2014–2017. Median age at diagnosis was 2 months (IQ 1–6 months). The main clinical findings were: bone alterations (59%); hepatosplenomegaly (54.1%); anemia (62.8%); skin lesions (42.6%) and renal compromise (33.3%). Cerebrospinal fluid (CSF) was abnormal in 5 patients, normal in 45 and was not available for 11 patients. Remarkably, spinal lumbar puncture did not modify therapeutic decisions in any case. Between mothers, only 46% have been tested for syphilis during pregnancy and 60.5% patients had non-treponemal titers equal to or less than fourfold the maternal titer. Intravenous penicillin G was prescribed for all except one patient, who received ceftriaxone with good therapeutic response. During follow-up, 1.6% infants died, 6.5% had persistent kidney disorders and 1.6% showed bone sequelae damage. RPR titers decreased after treatment, reaching negative seroconversion in 43% subjects at a median of 26.4 months. Low adherence to follow up was observed due to inherent vulnerable and low-income population characteristics in our cohort. Our results highlight a rising tendency in cases referred for CS in our population with high morbidity related to delayed diagnosis. A good therapeutic response was observed. CS requires a greater effort from the health system to adequately screen for this disease during pregnancy, and to detect cases earlier, to provide an adequate diagnosis and treatment.     

Larvicidal potential of cell wall degrading enzymes from Trichoderma asperellum against Aedes aegypti (Diptera: Culicidae)

Aedes aegypti is a mosquito vector of arboviruses such as dengue, chikungunya, zika and yellow fever that cause important public health diseases. The incidence and gravity of these diseases justifies the search for effective measures to reduce the presence of this vector in the environment. Bioinsecticides are an effective alternative method for insect control, with added ecological benefits such as biodegradability. The current study demonstrates that a chitinolytic enzyme complex produced by the fungus Trichoderma asperellum can disrupt cuticle formation in the L3 larvae phase of A. aegypti, suggesting such biolarvicidal action could be used for mosquito control. T. asperellum was exposed to chitin from different sources. This induction of cell wall degrading enzymes, including chitinase, N-acetylglucosaminidase and β-1,3-glucanase. Groups of 20 L3 larvae of A. aegypti were exposed to varying concentrations of chitinolytic enzymes induced with commercial chitin (CWDE) and larvae cell wall degrading enzymes (L-CWDE). After 72 h of exposure to the CWDE, 100% of larvae were killed. The same percent mortality was observed after 48 h of exposure to L-CWDE at half the CWDE enzyme mixture concentration. Exoskeleton deterioration was further observed by scanning and electron microscopy. Our findings indicate that L-CWDE produced by T. asperellum reflect chitinolytic enzymes with greater specificity for L3 larval biomolecules. This specificity is characterized by the high percentage of mortality compared with CWDE treatments and also by abrupt changes in patterns of the cellular structures visualized by scanning and transmission electron microscopy. These mixtures of chitinolytic enzymes could be candidates, as adjuvant or synergistic molecules, to replace conventional chemical insecticides currently in use.