Diabetic nephropathy(DN)is the most frequent cause of chronic renal failure.Until now,the pathophysiological mechanisms that determine its development and progression have not yet been elucidated.In the present study,...Diabetic nephropathy(DN)is the most frequent cause of chronic renal failure.Until now,the pathophysiological mechanisms that determine its development and progression have not yet been elucidated.In the present study,we evaluate the role of autophagy at early stages of DN,induced in type 2 diabetes mellitus(T2DM)mouse,and its association with proximal tubule membrane endocytic receptors,megalin and cubilin.In T2DM animals we observed a tubule-interstitial injury with significantly increased levels of urinary GGT and ALP,but an absence of tubulointerstitial fibrosis.Kidney proximal tubule cells of T2DM animals showed autophagic vesicles larger than those observed in the control group,and an increase in the number of these vesicles marked with LBPA by immunofluorescence.Furthermore,a significant decrease in the ratio of LC3II/LC3I isoforms and in p62 protein expression in DN affected animals is shown.Finally,we observed a marked increase in urinary albumin and vitamin D binding-protein levels in T2DM animals as well as a significant decrease in expression of megalin in the renal cortex.These results indicate an alteration of the tubular endocytic transporters in DN,which could be related to autophagic dysfunction,which would in turn result in impaired organelle recycling,thus contributing to the progression of this disease.展开更多
A decrease in the tubular expression of albumin endocytic transporters megalin and cubilin has been associated with diabetic nephropathy,but there are no comprehensive studies to date relating early tubulointerstitial...A decrease in the tubular expression of albumin endocytic transporters megalin and cubilin has been associated with diabetic nephropathy,but there are no comprehensive studies to date relating early tubulointerstitial injury and the effect of the disease on both transporters in type 1 diabetes mellitus(T1DM).We used eight-week-old male C57BL/6 mice divided into two groups;one of them received the vehicle(control group),while the other received the vehicle+200 mg/kg streptozotocin(T1DM).Ten weeks after the injection,we evaluated plasma insulin,enzymuria,urinary vitamin D-binding protein(VDBP),tubulointerstitial fibrosis and proximal tubule histology,markers of autophagy,and megalin and cubilin levels.We found a reduction in tubular protein reabsorption(albumin and VDBP as specific substances carried by both transporters)with increased tubulointerstitial injury,development of fibrosis,thickening of tubular basement membrane,and an increase in tubular cell metalloproteases.This was associated with a decrease in the renal expression of megalin and cubilin.We also observed an increase in the amount of cellular vesicles of the phagocytic system in the tubules,which could be linked to an alteration of normal intracellular trafficking of both receptors,thus affecting the normal function of transporters in early stages of diabetic nephropathy.In diabetic animals,the added effects of tubulointerstitial injury,the decreases in megalin and cubilin expression,and an altered intracellular trafficking of these receptors,seriously affect protein reabsorption.展开更多
<b>Introduction:</b> <i>Burkholderia cepacia</i> is a non-fermenting emergent bacterium common in nosocomial infections and can cause life-threatening infections whose multidrug resistance make...<b>Introduction:</b> <i>Burkholderia cepacia</i> is a non-fermenting emergent bacterium common in nosocomial infections and can cause life-threatening infections whose multidrug resistance makes them a serious threat in hospitals. The aim of this study was to determine the prevalence of <i>B. cepacia</i> infections during nosocomial infections at Libreville University teaching hospital. <b>Methodology:</b> In this cross-sectional study, lasting 19 months, 412 blood cultures were analyzed. The BacT/ALERT 3D (Biomerieux, France) was used to detect the positivity of blood culture flasks and the Viteck 2 compact (Biomerieux, France) for the identification of germs and the study of their susceptibility to antibiotics. <b>Results:</b> Our study population consisted of 412 patients. The sex-ratio M/F was 1.06 in favor of the male gender (n = 201, 51%). The age of the patients varied between 0 and 82 years. The bacteremia of <i>B. cepacia</i> mainly affected children under 15 years of age with a prevalence of 7% (n = 28). The pediatric ward was more represented with a frequency of 36% (n = 10). The antibiotic sensitivity profile showed high resistance of 100% for aminoglycosides (amikacin, tobramycin, and gentamycin), tetracycline, beta-lactams (Amoxicillin, Imipenem, Ticarcillin, Cefoxitin and Cefotaxime), and ciprofloxacin. However, four molecules were active on <i>B. cepacia</i> (Levofloxacin 100%, Trimethoprim + sulfamethoxazole 92.3%, ceftazidime 80% and cefepime 35%). <b>Conclusion:</b> Ultimately, infection and multi-resistance due to <i>Burkholderia cepacia</i> calls for a review of hospital hygiene in the pediatric ward and a review of antibiotic therapy in young children.展开更多
Spinal muscular atrophy(SMA)is caused by dysfunction of the alpha motor neurons of the spinal cord.It is an autosomal recessive disease associated to the SMN1 gene,located in the subtelomeric region of 5q13.A paralog ...Spinal muscular atrophy(SMA)is caused by dysfunction of the alpha motor neurons of the spinal cord.It is an autosomal recessive disease associated to the SMN1 gene,located in the subtelomeric region of 5q13.A paralog SMN2 gene is located at the centromeric region of the same chromosome,which apparently originated by an ancestral inverted duplication occurring only in humans.The exon sequence differs in two nucleotides in exon 7 and exon 8,which leads to an SMN2 transcript that lacks exon 7 and results in a truncated protein.Part(10%)of the SMN2 transcripts avoids the splicing of exon 7 but most of the copies are dysfunctional.In a disease scenario,the more SMN2 copies the higher possibility to restore at least partly the effects of SMN1 deficiency.Some therapeutic approaches are being developed to increase the expression of SMN2.To determine the number of SMN1 and SMN2 copies,the methodology must distinguish accurately between both genes.In this work,we present the results obtained using multiplex ligation-dependent probe amplification(MLPA)in 60 SMA suspected patients/carriers derived from different regions of Argentina.In 32 of these DNA samples we found alterations in SMN1.Among these,16 presented a heterozygous deletion(carrier status)and 14 an homozygous deletion(patient status)in exon 7 and 8 of SMN1.In one case,exon 7 was found homozygously deleted but exon 8 presented a single copy,and in another case,exon 7 was found heterozygously deleted while exon 8 was normal.Almost half of the patients(7/15)presented a normal diploid number of SMN2 while the other half(8/15)presented an increased number.In this work we showed how a probe-based methodology such as MLPA was able to distinguish between the paralog genes and determine the amount of copies in DNA samples from suspected patients/carriers of SMA.展开更多
Trypanosoma cruzi is the causative agent of Chagas disease.This parasite requires the intracellular niche in order to proliferate and disseminate the infection.After invasion,T.cruzi resides temporarily in an acidic v...Trypanosoma cruzi is the causative agent of Chagas disease.This parasite requires the intracellular niche in order to proliferate and disseminate the infection.After invasion,T.cruzi resides temporarily in an acidic vacuole which is lysed by a not well-understood mechanism.Transmission electron microscopy was used to describe the process of T.cruzi escape from the parasitophorous vacuole over the time.Using HeLa(non-professional phagocytic cells)as host cell,we observed that recently internalized parasites reside in a membrane-bounded vacuole.A few hours later,the first sign of vacuole disruption appeared as membrane discontinuities.This observation was followed by a progressive vacuole swelling as evidenced by an electron-lucent halo between the parasite and the vacuole membrane.Apparently,the vacuole membrane remnants reorganized as small vesicles that eventually disappeared from the vicinity of the parasites.Finally,parasites reach the host cell cytosol where replication takes place.The thorough ultrastructural description of this process set the base for a comprehensive understanding of the parasite-host cell interaction and,thus open the possibility of new therapeutic intervention strategies.展开更多
The digestive gland of the apple snail Pomacea canaliculata lodges two types of pigmented corpuscles(identified as C and K corpuscles)which has been proposed as endosymbiont/s.Both corpuscular types are always present...The digestive gland of the apple snail Pomacea canaliculata lodges two types of pigmented corpuscles(identified as C and K corpuscles)which has been proposed as endosymbiont/s.Both corpuscular types are always present in the digestive gland of adult snails,they are released into the tubuloacinar lumen and are later expelled in the feces.On their part,hatchlings lack any C or K corpuscles in the digestive gland as well as in their feces,whereas C corpuscles appear in both the gland and feces within one week after hatching.Hence,it is possible that the detritivorous hatchlings acquire the putative C-endosymbiont from feces in the sediments where they live,i.e.through‘lateral’or‘horizontal’transmission.This possibility was put to test in an experiment in which we prevented any lateral transmission,by a 7-days aseptic culture,with no food,of aseptically obtained hatchlings.At the end of the experiment,we observed that most juveniles had survived the culture period,and hence the digestive glands and feces of survivors were studied by light microscopy of resin embedded,toluidine blue-stained sections.All studied glands and fecal samples showed C corpuscles.It is concluded that lateral transmission of the endosymbiont,if any,is not indispensable for the acquisition of the endosymbiont by hatchlings.展开更多
The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with...The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment.In particular,proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes.In this review,the potential of enhanced molecular dynamics to address cell-biology problems is discussed.The cases of membrane deformation,membrane fusion,and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables.Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented.Finally,novel strategies merging cell biology and physics are considered as future lines of research.展开更多
Cells are open systems that exchange energy and molecules with their environment.As any material system,they perform all the complex activities required for homeostasis and reproduction,obeying the thermodynamic laws....Cells are open systems that exchange energy and molecules with their environment.As any material system,they perform all the complex activities required for homeostasis and reproduction,obeying the thermodynamic laws.This viewpoint will argue that the basic logic governing the energy flux required to preserve cell organization and function is simple:to decrease the activation energy(Ea)of specific processes.Almost none of the possible chemical reactions and energy transformations inside a cell occur at a measurable speed at room or body temperature.Enzymes or other macromolecular structures speed up particular transformations by decreasing the corresponding energetic barriers.However,to maintain the systems in a homeostatic state,capable of sophisticated functions based on this simple strategy requires an inconceivably complex solution.The conclusion will point to the challenging and intricate problems that cells have solved to carve the highly regulated channel through which the energy flows,fueling the work of these nanoscale machines.展开更多
Until recently,microglia were mainly known as the resident phagocytes of the brain,i.e.the‘immunological warriors’of the brain.However,extensive knowledge is being accumulated about the functions of microglia beyond...Until recently,microglia were mainly known as the resident phagocytes of the brain,i.e.the‘immunological warriors’of the brain.However,extensive knowledge is being accumulated about the functions of microglia beyond immunity.Nowadays,it is well accepted that microglial cells are highly dynamic and responsive,and that they intervene in a dual manner in many developmental processes that shape the central nervous system,including neurogenesis,gliogenesis,spatial patterning,synaptic formation and elimination,and neural circuit establishment and maturation.The differentiation and the pool of precursor cells were also shown to be under microglia regulation via bidirectional communication.In this concise review,I discuss our recent work in microglia-Pax6+cell interactions in one of the circumventricular organs,the pineal gland.An analogy with the rest of the central nervous system is also presented.In addition,I briefly examine mechanisms of interaction between microglia and non-microglial cells in both health and disease.New avenues are also introduced,which may lead us to better comprehend the impact of microglia in physiological and pathological conditions.展开更多
Trypanosoma rangeli and T.cruzi are both parasitic unicellular species that infect humans.Unlike T.cruzi,the causative agent of Chagas disease,T.rangeli is an infective and non-pathogenic parasite for humans,but patho...Trypanosoma rangeli and T.cruzi are both parasitic unicellular species that infect humans.Unlike T.cruzi,the causative agent of Chagas disease,T.rangeli is an infective and non-pathogenic parasite for humans,but pathogenic for vectors from the Rhodnius genus.Because both species can coexist in different hosts and overlap their infective cycles but very little is known about the infection of T.rangeli in mammalian cells,we decided to characterize both the development of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action on it.We found that T.rangeli exhibits a cycle of infection in Vero cells similar to that for T.cruzi and that the repurposed drug,17-AAG,and the natural extract Artemisia sp.essential oil produce a toxic effect on epimastigotes showing a trypanocidal action from the fifth day of culture.Both treatments also affected the infection of trypomastigotes and reduced the capacity of replication of amastigotes of T.rangeli.Since T.cruzi/T.rangeli coinfection cases have been reported,the finding of drugs with potential activity against both species could be significant in the future.Furthermore,studies of susceptibility of both species to drugs could also help to know the different mechanisms of pathogenicity in humans displayed by T.cruzi that are absent in T.rangeli.展开更多
Coxiella burnetii is an obligate intracellular pathogen and the causative agent of Q fever.In this brief review,we describe how recently described mechanisms help our understanding of C.burnetii invasion and its survi...Coxiella burnetii is an obligate intracellular pathogen and the causative agent of Q fever.In this brief review,we describe how recently described mechanisms help our understanding of C.burnetii invasion and its survival in the host cell by the formation of a replicative niche:the Coxiella-containing vacuole.We describe the actin-associated proteins involved in the internalization of C.burnetii,and we discuss the contribution of diverse degradation pathways of the cell during the formation and stabilization of the Coxiella-containing vacuole.展开更多
Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical charact...Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabiUzed human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranyigeranyiated, and active Rab3A elicited human sperm exocytosis perse. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino.terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.展开更多
Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; ho...Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without （untreated control） 3-morpholinosydnonimine （SIN-l）, in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition （MPT）, externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-l, the sperm viability was significantly reduced compared to untreated control （P〈 0.001）. Furthermore, the MPT was induced （P〈 0.01） and increment in DNA oxidation （P 〈 0.01）, DNA fragmentation （P 〈 0.01）, tyrosine nitration （P 〈 0.0001） and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control （P 〈 0.001）, this process was observed in 〈10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.展开更多
Autophagy is an essential cellular homeostatic mechanism by which intracellular components are delivered into the lysosomes for degradation and recycling.Autophagy has been related with a diversity of pathological or ...Autophagy is an essential cellular homeostatic mechanism by which intracellular components are delivered into the lysosomes for degradation and recycling.Autophagy has been related with a diversity of pathological or physiological dentary processes such as bone remodeling,skeletal aging,osteoclastogenesis,osteoblastogenesis and different types of oral cancer.Platelet-rich plasma(PRP),isolated from autologous blood,is a plasma preparation containing a higher concentration of platelets which contains numerous different growth factors and cytokines that activate several cellular signaling cascades.The purpose of this study is to investigate the effect of PRP on autophagy stimulation in both osteoblast precursor 3T3-L1 and non-related osteoblastic cells.Our results showed that PRP can increase the number of autophagic structures in 3T3-L1 and HeLa(cervical cancer cells)cells.Moreover,we have determined by Western blot a rise in the lipidated form of the autophagic protein LC3(i.e.LC3-II)upon PRP treatment.Taken together,our results suggest that PRP is able to induce a strongly autophagy response in osteoblast precursor and,to a lesser extent,in non-related osteoblastic cells,suggesting that PRP could be a potential therapeutic tool for some autophagy-related diseases associated with bone homeostasis.展开更多
Pigments present in the brown-greenish C morph of an intracellular endosymbiont of Pomacea canaliculata were investigated.Acetone extracts of the endosymbiotic corpuscles showed an absorption spectrum similar to that ...Pigments present in the brown-greenish C morph of an intracellular endosymbiont of Pomacea canaliculata were investigated.Acetone extracts of the endosymbiotic corpuscles showed an absorption spectrum similar to that of chlorophylls.Three fractions obtained from silica gel column chromatography of the acetone extracts(CI,CII and CIII),were studied by positive ion fast atom bombardment-mass spectrometry(FAB–MS)and hydrogen-nuclear magnetic resonance(H-NMR).Results indicated the presence of(1)a sterol in the yellow colored CI fraction;(2)a mixture of pheophorbides a and b in the major green fraction,CII;and(3)a modified pheophorbide a in the smaller green fraction,CIII.Aqueous extracts of the C endosymbiont did not show evidence of the occurrence of C-phycocyanin,allophycocyanin or phycoerithrin(light absorption,fluorescence emission,and electrophoresis of the protein moieties)while cyanobacterial cells(Nostoc sp.)showed evidence of C-phycocyanin and allophycocyanin.The possible phylogenetic and functional significance of the pigments present in the C endosymbiont is discussed.展开更多
In the last years,much work has shown that the most effective repair system of the body is represented by stem cells,which are defined as undifferentiated precursors that own unlimited or prolonged self-renewal abilit...In the last years,much work has shown that the most effective repair system of the body is represented by stem cells,which are defined as undifferentiated precursors that own unlimited or prolonged self-renewal ability,which also have the potential to transform themselves into various cell types through differentiation.All tissues that form the body contain many different types of somatic cells,along with stem cells that are called‘mesenchymal stem(or stromal)cells’(MSC).In certain circumstances,some of these MSC migrate to injured tissues to replace dead cells or to undergo differentiation to repair it.The discovery of MSC has been an important step in regenerative medicine because of their high versatility.Moreover,the finding of a method to isolate MSC from adipose tissue,so called‘adipose-derived mesenchymal stem cells’(ASC),which share similar differentiation capabilities and isolation yield that is greater than other MSC,and less bioethical concerns compared to embryonic stem cells,have created self-praised publicity to procure almost any treatment with them.Here,we review the current techniques for isolation,culture and differentiation of human ASC(hASC),and describe them in detail.We also compile some advantages of the hASC over other stem cells,and provide some concepts that could help finding strategies to promote their therapeutic efficiency.展开更多
In this study,we propose an automatic procedure for digital image processing.We describe a method that can efficiently quantify and characterize lipid droplets distributions in different cell types in culture.Prospect...In this study,we propose an automatic procedure for digital image processing.We describe a method that can efficiently quantify and characterize lipid droplets distributions in different cell types in culture.Prospectively,the lipid droplets detection method described in this work could be applied to static or time-lapse data,collected with a simple visible light or fluorescence microscopy equipment.Fully automated algorithms were implemented in Octave,a freely available scientific package.展开更多
The testis is a double gland comprised of the sperm-producing seminiferous tubules and a complex endocrine interstice.The former structures generate and release whole cells(exocrine aspect of the gland)while the latte...The testis is a double gland comprised of the sperm-producing seminiferous tubules and a complex endocrine interstice.The former structures generate and release whole cells(exocrine aspect of the gland)while the latter synthesize and release androgens and related hormones.The testis also has poorly understood paracrine connexions.These connexions play an important role during spermatogenesis.A key molecule within these structures is β-actin.Therefore,the present study aims at examining the expression pattern of β-actin during the various stages of the spermatic cycle in the rat.To achieve this goal,we used a combination of trans-illumination assisted microdissection of seminiferous tubules,confocal immunofluorescence and Western blot analysis.Our experiments revealed that the levels of β-actin fluctuate significantly during spermatogenesis,peaking immediately after spermiation.展开更多
基金supported by grants from FONDECyT PD2014[3140024]Fundación Florencio Fiorini(Beca Estímulo para Investigación en Medicina 2016)to M.Giraud-Billoud.
文摘Diabetic nephropathy(DN)is the most frequent cause of chronic renal failure.Until now,the pathophysiological mechanisms that determine its development and progression have not yet been elucidated.In the present study,we evaluate the role of autophagy at early stages of DN,induced in type 2 diabetes mellitus(T2DM)mouse,and its association with proximal tubule membrane endocytic receptors,megalin and cubilin.In T2DM animals we observed a tubule-interstitial injury with significantly increased levels of urinary GGT and ALP,but an absence of tubulointerstitial fibrosis.Kidney proximal tubule cells of T2DM animals showed autophagic vesicles larger than those observed in the control group,and an increase in the number of these vesicles marked with LBPA by immunofluorescence.Furthermore,a significant decrease in the ratio of LC3II/LC3I isoforms and in p62 protein expression in DN affected animals is shown.Finally,we observed a marked increase in urinary albumin and vitamin D binding-protein levels in T2DM animals as well as a significant decrease in expression of megalin in the renal cortex.These results indicate an alteration of the tubular endocytic transporters in DN,which could be related to autophagic dysfunction,which would in turn result in impaired organelle recycling,thus contributing to the progression of this disease.
基金supported by Grant FONDECyT PD2014[3140024]to Maximiliano Giraud-Billoud.
文摘A decrease in the tubular expression of albumin endocytic transporters megalin and cubilin has been associated with diabetic nephropathy,but there are no comprehensive studies to date relating early tubulointerstitial injury and the effect of the disease on both transporters in type 1 diabetes mellitus(T1DM).We used eight-week-old male C57BL/6 mice divided into two groups;one of them received the vehicle(control group),while the other received the vehicle+200 mg/kg streptozotocin(T1DM).Ten weeks after the injection,we evaluated plasma insulin,enzymuria,urinary vitamin D-binding protein(VDBP),tubulointerstitial fibrosis and proximal tubule histology,markers of autophagy,and megalin and cubilin levels.We found a reduction in tubular protein reabsorption(albumin and VDBP as specific substances carried by both transporters)with increased tubulointerstitial injury,development of fibrosis,thickening of tubular basement membrane,and an increase in tubular cell metalloproteases.This was associated with a decrease in the renal expression of megalin and cubilin.We also observed an increase in the amount of cellular vesicles of the phagocytic system in the tubules,which could be linked to an alteration of normal intracellular trafficking of both receptors,thus affecting the normal function of transporters in early stages of diabetic nephropathy.In diabetic animals,the added effects of tubulointerstitial injury,the decreases in megalin and cubilin expression,and an altered intracellular trafficking of these receptors,seriously affect protein reabsorption.
文摘<b>Introduction:</b> <i>Burkholderia cepacia</i> is a non-fermenting emergent bacterium common in nosocomial infections and can cause life-threatening infections whose multidrug resistance makes them a serious threat in hospitals. The aim of this study was to determine the prevalence of <i>B. cepacia</i> infections during nosocomial infections at Libreville University teaching hospital. <b>Methodology:</b> In this cross-sectional study, lasting 19 months, 412 blood cultures were analyzed. The BacT/ALERT 3D (Biomerieux, France) was used to detect the positivity of blood culture flasks and the Viteck 2 compact (Biomerieux, France) for the identification of germs and the study of their susceptibility to antibiotics. <b>Results:</b> Our study population consisted of 412 patients. The sex-ratio M/F was 1.06 in favor of the male gender (n = 201, 51%). The age of the patients varied between 0 and 82 years. The bacteremia of <i>B. cepacia</i> mainly affected children under 15 years of age with a prevalence of 7% (n = 28). The pediatric ward was more represented with a frequency of 36% (n = 10). The antibiotic sensitivity profile showed high resistance of 100% for aminoglycosides (amikacin, tobramycin, and gentamycin), tetracycline, beta-lactams (Amoxicillin, Imipenem, Ticarcillin, Cefoxitin and Cefotaxime), and ciprofloxacin. However, four molecules were active on <i>B. cepacia</i> (Levofloxacin 100%, Trimethoprim + sulfamethoxazole 92.3%, ceftazidime 80% and cefepime 35%). <b>Conclusion:</b> Ultimately, infection and multi-resistance due to <i>Burkholderia cepacia</i> calls for a review of hospital hygiene in the pediatric ward and a review of antibiotic therapy in young children.
文摘Spinal muscular atrophy(SMA)is caused by dysfunction of the alpha motor neurons of the spinal cord.It is an autosomal recessive disease associated to the SMN1 gene,located in the subtelomeric region of 5q13.A paralog SMN2 gene is located at the centromeric region of the same chromosome,which apparently originated by an ancestral inverted duplication occurring only in humans.The exon sequence differs in two nucleotides in exon 7 and exon 8,which leads to an SMN2 transcript that lacks exon 7 and results in a truncated protein.Part(10%)of the SMN2 transcripts avoids the splicing of exon 7 but most of the copies are dysfunctional.In a disease scenario,the more SMN2 copies the higher possibility to restore at least partly the effects of SMN1 deficiency.Some therapeutic approaches are being developed to increase the expression of SMN2.To determine the number of SMN1 and SMN2 copies,the methodology must distinguish accurately between both genes.In this work,we present the results obtained using multiplex ligation-dependent probe amplification(MLPA)in 60 SMA suspected patients/carriers derived from different regions of Argentina.In 32 of these DNA samples we found alterations in SMN1.Among these,16 presented a heterozygous deletion(carrier status)and 14 an homozygous deletion(patient status)in exon 7 and 8 of SMN1.In one case,exon 7 was found homozygously deleted but exon 8 presented a single copy,and in another case,exon 7 was found heterozygously deleted while exon 8 was normal.Almost half of the patients(7/15)presented a normal diploid number of SMN2 while the other half(8/15)presented an increased number.In this work we showed how a probe-based methodology such as MLPA was able to distinguish between the paralog genes and determine the amount of copies in DNA samples from suspected patients/carriers of SMA.
基金This work was financed by grants from Universidad Nacional de Cuyo to JAC and PSR(J043 and J481)Agencia Nacional de Promoción Científica y Tecnológica PICT 2013-2757 to PSR.
文摘Trypanosoma cruzi is the causative agent of Chagas disease.This parasite requires the intracellular niche in order to proliferate and disseminate the infection.After invasion,T.cruzi resides temporarily in an acidic vacuole which is lysed by a not well-understood mechanism.Transmission electron microscopy was used to describe the process of T.cruzi escape from the parasitophorous vacuole over the time.Using HeLa(non-professional phagocytic cells)as host cell,we observed that recently internalized parasites reside in a membrane-bounded vacuole.A few hours later,the first sign of vacuole disruption appeared as membrane discontinuities.This observation was followed by a progressive vacuole swelling as evidenced by an electron-lucent halo between the parasite and the vacuole membrane.Apparently,the vacuole membrane remnants reorganized as small vesicles that eventually disappeared from the vicinity of the parasites.Finally,parasites reach the host cell cytosol where replication takes place.The thorough ultrastructural description of this process set the base for a comprehensive understanding of the parasite-host cell interaction and,thus open the possibility of new therapeutic intervention strategies.
文摘The digestive gland of the apple snail Pomacea canaliculata lodges two types of pigmented corpuscles(identified as C and K corpuscles)which has been proposed as endosymbiont/s.Both corpuscular types are always present in the digestive gland of adult snails,they are released into the tubuloacinar lumen and are later expelled in the feces.On their part,hatchlings lack any C or K corpuscles in the digestive gland as well as in their feces,whereas C corpuscles appear in both the gland and feces within one week after hatching.Hence,it is possible that the detritivorous hatchlings acquire the putative C-endosymbiont from feces in the sediments where they live,i.e.through‘lateral’or‘horizontal’transmission.This possibility was put to test in an experiment in which we prevented any lateral transmission,by a 7-days aseptic culture,with no food,of aseptically obtained hatchlings.At the end of the experiment,we observed that most juveniles had survived the culture period,and hence the digestive glands and feces of survivors were studied by light microscopy of resin embedded,toluidine blue-stained sections.All studied glands and fecal samples showed C corpuscles.It is concluded that lateral transmission of the endosymbiont,if any,is not indispensable for the acquisition of the endosymbiont by hatchlings.
基金Grants from CONICET(PIP-0409CO)ANPCyT(PICT2020-1897)are gratefully acknowledged。
文摘The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment.In particular,proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes.In this review,the potential of enhanced molecular dynamics to address cell-biology problems is discussed.The cases of membrane deformation,membrane fusion,and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables.Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented.Finally,novel strategies merging cell biology and physics are considered as future lines of research.
文摘Cells are open systems that exchange energy and molecules with their environment.As any material system,they perform all the complex activities required for homeostasis and reproduction,obeying the thermodynamic laws.This viewpoint will argue that the basic logic governing the energy flux required to preserve cell organization and function is simple:to decrease the activation energy(Ea)of specific processes.Almost none of the possible chemical reactions and energy transformations inside a cell occur at a measurable speed at room or body temperature.Enzymes or other macromolecular structures speed up particular transformations by decreasing the corresponding energetic barriers.However,to maintain the systems in a homeostatic state,capable of sophisticated functions based on this simple strategy requires an inconceivably complex solution.The conclusion will point to the challenging and intricate problems that cells have solved to carve the highly regulated channel through which the energy flows,fueling the work of these nanoscale machines.
基金Supported by grants from CONICET(Argentina,EM,PIP-CONICET 112-201101-00247,http://www.conicet.gov.ar)ANPCyT(Argentina,EM,PICT 2012-174,PICT 2017-499+1 种基金http://www.agencia.mincyt.gob.ar)NIH-CONICET(Argentina and USA,EM and Stephen Noctor,F65096).
文摘Until recently,microglia were mainly known as the resident phagocytes of the brain,i.e.the‘immunological warriors’of the brain.However,extensive knowledge is being accumulated about the functions of microglia beyond immunity.Nowadays,it is well accepted that microglial cells are highly dynamic and responsive,and that they intervene in a dual manner in many developmental processes that shape the central nervous system,including neurogenesis,gliogenesis,spatial patterning,synaptic formation and elimination,and neural circuit establishment and maturation.The differentiation and the pool of precursor cells were also shown to be under microglia regulation via bidirectional communication.In this concise review,I discuss our recent work in microglia-Pax6+cell interactions in one of the circumventricular organs,the pineal gland.An analogy with the rest of the central nervous system is also presented.In addition,I briefly examine mechanisms of interaction between microglia and non-microglial cells in both health and disease.New avenues are also introduced,which may lead us to better comprehend the impact of microglia in physiological and pathological conditions.
文摘Trypanosoma rangeli and T.cruzi are both parasitic unicellular species that infect humans.Unlike T.cruzi,the causative agent of Chagas disease,T.rangeli is an infective and non-pathogenic parasite for humans,but pathogenic for vectors from the Rhodnius genus.Because both species can coexist in different hosts and overlap their infective cycles but very little is known about the infection of T.rangeli in mammalian cells,we decided to characterize both the development of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action on it.We found that T.rangeli exhibits a cycle of infection in Vero cells similar to that for T.cruzi and that the repurposed drug,17-AAG,and the natural extract Artemisia sp.essential oil produce a toxic effect on epimastigotes showing a trypanocidal action from the fifth day of culture.Both treatments also affected the infection of trypomastigotes and reduced the capacity of replication of amastigotes of T.rangeli.Since T.cruzi/T.rangeli coinfection cases have been reported,the finding of drugs with potential activity against both species could be significant in the future.Furthermore,studies of susceptibility of both species to drugs could also help to know the different mechanisms of pathogenicity in humans displayed by T.cruzi that are absent in T.rangeli.
基金supported by grants from Agencia Nacional de Promocion Cientifica y Tecnologica(PICT2012 no.2425)SECTyP-Universidad Nacional de Cuyo(06/J468)to W.B.
文摘Coxiella burnetii is an obligate intracellular pathogen and the causative agent of Q fever.In this brief review,we describe how recently described mechanisms help our understanding of C.burnetii invasion and its survival in the host cell by the formation of a replicative niche:the Coxiella-containing vacuole.We describe the actin-associated proteins involved in the internalization of C.burnetii,and we discuss the contribution of diverse degradation pathways of the cell during the formation and stabilization of the Coxiella-containing vacuole.
文摘Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabiUzed human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranyigeranyiated, and active Rab3A elicited human sperm exocytosis perse. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino.terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
文摘Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without （untreated control） 3-morpholinosydnonimine （SIN-l）, in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition （MPT）, externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-l, the sperm viability was significantly reduced compared to untreated control （P〈 0.001）. Furthermore, the MPT was induced （P〈 0.01） and increment in DNA oxidation （P 〈 0.01）, DNA fragmentation （P 〈 0.01）, tyrosine nitration （P 〈 0.0001） and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control （P 〈 0.001）, this process was observed in 〈10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
基金partly supported by grants from Agencia Nacional de Promoción Científica y Tecnológica(PICT 2013-2335)SeCTyP K013(Universidad Nacional de Cuyo)to Claudio M.Fader.
文摘Autophagy is an essential cellular homeostatic mechanism by which intracellular components are delivered into the lysosomes for degradation and recycling.Autophagy has been related with a diversity of pathological or physiological dentary processes such as bone remodeling,skeletal aging,osteoclastogenesis,osteoblastogenesis and different types of oral cancer.Platelet-rich plasma(PRP),isolated from autologous blood,is a plasma preparation containing a higher concentration of platelets which contains numerous different growth factors and cytokines that activate several cellular signaling cascades.The purpose of this study is to investigate the effect of PRP on autophagy stimulation in both osteoblast precursor 3T3-L1 and non-related osteoblastic cells.Our results showed that PRP can increase the number of autophagic structures in 3T3-L1 and HeLa(cervical cancer cells)cells.Moreover,we have determined by Western blot a rise in the lipidated form of the autophagic protein LC3(i.e.LC3-II)upon PRP treatment.Taken together,our results suggest that PRP is able to induce a strongly autophagy response in osteoblast precursor and,to a lesser extent,in non-related osteoblastic cells,suggesting that PRP could be a potential therapeutic tool for some autophagy-related diseases associated with bone homeostasis.
基金supported by grants from FONCyT,CONICET and the National University of Cuyo.
文摘Pigments present in the brown-greenish C morph of an intracellular endosymbiont of Pomacea canaliculata were investigated.Acetone extracts of the endosymbiotic corpuscles showed an absorption spectrum similar to that of chlorophylls.Three fractions obtained from silica gel column chromatography of the acetone extracts(CI,CII and CIII),were studied by positive ion fast atom bombardment-mass spectrometry(FAB–MS)and hydrogen-nuclear magnetic resonance(H-NMR).Results indicated the presence of(1)a sterol in the yellow colored CI fraction;(2)a mixture of pheophorbides a and b in the major green fraction,CII;and(3)a modified pheophorbide a in the smaller green fraction,CIII.Aqueous extracts of the C endosymbiont did not show evidence of the occurrence of C-phycocyanin,allophycocyanin or phycoerithrin(light absorption,fluorescence emission,and electrophoresis of the protein moieties)while cyanobacterial cells(Nostoc sp.)showed evidence of C-phycocyanin and allophycocyanin.The possible phylogenetic and functional significance of the pigments present in the C endosymbiont is discussed.
基金supported by grants from ANPCyT(PICT-141659)CONICET(PIP-13CO01),SeCTyP-UNCuyo(J051)the Roemmers Foundation.DM,MU and DMB are members of CONICET.YRFL and ADG,are fellows of the same institution.
文摘In the last years,much work has shown that the most effective repair system of the body is represented by stem cells,which are defined as undifferentiated precursors that own unlimited or prolonged self-renewal ability,which also have the potential to transform themselves into various cell types through differentiation.All tissues that form the body contain many different types of somatic cells,along with stem cells that are called‘mesenchymal stem(or stromal)cells’(MSC).In certain circumstances,some of these MSC migrate to injured tissues to replace dead cells or to undergo differentiation to repair it.The discovery of MSC has been an important step in regenerative medicine because of their high versatility.Moreover,the finding of a method to isolate MSC from adipose tissue,so called‘adipose-derived mesenchymal stem cells’(ASC),which share similar differentiation capabilities and isolation yield that is greater than other MSC,and less bioethical concerns compared to embryonic stem cells,have created self-praised publicity to procure almost any treatment with them.Here,we review the current techniques for isolation,culture and differentiation of human ASC(hASC),and describe them in detail.We also compile some advantages of the hASC over other stem cells,and provide some concepts that could help finding strategies to promote their therapeutic efficiency.
基金supported by grants from ANPCyT(PICT-111450,PICT-122498 and PICT-141659)MinCyT(Iniciativa de Proyectos Acelerados de C\’alculo)IPAC 2016(SACT 017-00721036)+3 种基金IPAC 2017(2017-75-APN-SECACT\#MCT)CONICET(PIP-13CO01)SeCTyP-UNCuyo(J051)the Roemmers Foundation.
文摘In this study,we propose an automatic procedure for digital image processing.We describe a method that can efficiently quantify and characterize lipid droplets distributions in different cell types in culture.Prospectively,the lipid droplets detection method described in this work could be applied to static or time-lapse data,collected with a simple visible light or fluorescence microscopy equipment.Fully automated algorithms were implemented in Octave,a freely available scientific package.
文摘The testis is a double gland comprised of the sperm-producing seminiferous tubules and a complex endocrine interstice.The former structures generate and release whole cells(exocrine aspect of the gland)while the latter synthesize and release androgens and related hormones.The testis also has poorly understood paracrine connexions.These connexions play an important role during spermatogenesis.A key molecule within these structures is β-actin.Therefore,the present study aims at examining the expression pattern of β-actin during the various stages of the spermatic cycle in the rat.To achieve this goal,we used a combination of trans-illumination assisted microdissection of seminiferous tubules,confocal immunofluorescence and Western blot analysis.Our experiments revealed that the levels of β-actin fluctuate significantly during spermatogenesis,peaking immediately after spermiation.
