VEGF, but Not BDNF, Prevents the Downregulation of KCC2 Induced by Axotomy in Extraocular Motoneurons.

The potassium-chloride cotransporter KCC2 is the main extruder of Cl- in neurons. It plays a fundamental role in the activity of the inhibitory neurotransmitters (GABA and glycine) since low levels of KCC2 promote intracellular Cl- accumulation, leading to the depolarizing activity of GABA and glycine. The downregulation of this cotransporter occurs in neurological disorders characterized by hyperexcitability, such as epilepsy, neuropathic pain, and spasticity. KCC2 is also downregulated after axotomy. If muscle reinnervation is allowed, the KCC2 levels recover in motoneurons. Therefore, we argued that target-derived neurotrophic factors might be involved in the regulation of KCC2 expression. For this purpose, we performed the axotomy of extraocular motoneurons via the monocular enucleation of adult rats, and a pellet containing either VEGF or BDNF was chronically implanted in the orbit. Double confocal immunofluorescence of choline acetyl-transferase (ChAT) and KCC2 was carried out in the brainstem sections. Axotomy led to a KCC2 decrease in the neuropil and somata of extraocular motoneurons, peaking at 15 days post-lesion, with the exception of the abducens motoneuron somata. VEGF administration prevented the axotomy-induced KCC2 downregulation. By contrast, BDNF either maintained or reduced the KCC2 levels following axotomy, suggesting that BDNF is involved in the axotomy-induced KCC2 downregulation in extraocular motoneurons. The finding that VEGF prevents KCC2 decrease opens up new possibilities for the treatment of neurological disorders coursing with neuronal hyperactivity due to KCC2 downregulation.

Relative contribution of lateral vestibular neuron and abducens internuclear neuron inputs to the discharge activity of medial rectus motoneurons.

Medial rectus motoneurons mediate nasally directed horizontal eye movements. These motoneurons receive two major excitatory inputs, from the abducens internuclear neurons (ABD Ints) and neurons of the lateral vestibular nucleus whose axons course through the ascending tract of Deiters (ATD). In the present work, we have recorded in the alert chronic cat preparation the discharge activity of these two premotor neurons simultaneously with eye movements, to discern their relative contribution to the firing pattern of medial rectus motoneurons. ABD Int discharge was accurately correlated with eye movements, displaying high sensitivities to eye position and eye velocity. ATD neurons also discharged in relation to spontaneous and vestibular eye movements but showed significantly lower eye position and eye velocity sensitivities. Outstandingly, ATD neurons presented a significantly lower eye position threshold for recruitment compared to both ABD Ints and medial rectus motoneurons. Therefore, ATD neurons exhibited eye position and velocity signals during spontaneous and vestibular eye movements, which were of lower magnitude than those of ABD Ints, but due to their low recruitment threshold, they could play a significant role in facilitating ABD Int signal transmission onto medial rectus motoneurons.

Proprioceptors in extraocular muscles.

Proprioception is the sense that lets us perceive the location, movement and action of the body parts. The proprioceptive apparatus includes specialized sense organs (proprioceptors) which are embedded in the skeletal muscles. The eyeballs are moved by six pairs of eye muscles and binocular vision depends on fine-tuned coordination of the optical axes of both eyes. Although experimental studies indicate that the brain has access to eye position information, both classical proprioceptors (muscle spindles and Golgi tendon organ) are absent in the extraocular muscles of most mammalian species. This paradox of monitoring extraocular muscle activity in the absence of typical proprioceptors seemed to be resolved when a particular nerve specialization (the palisade ending) was detected in the extraocular muscles of mammals. In fact, for decades there was consensus that palisade endings were sensory structures that provide eye position information. The sensory function was called into question when recent studies revealed the molecular phenotype and the origin of palisade endings. Today we are faced with the fact that palisade endings exhibit sensory as well as motor features. This review aims to evaluate the literature on extraocular muscle proprioceptors and palisade endings and to reconsider current knowledge of their structure and function.

Novel Aspects of cAMP-Response Element Modulator (CREM) Role in Spermatogenesis and Male Fertility.

Spermatogenesis is a very complex process with an intricate transcriptional regulation. The transition from the diploid to the haploid state requires the involvement of specialized genes in meiosis, among other specific functions for the formation of the spermatozoon. The transcription factor cAMP-response element modulator (CREM) is a key modulator that triggers the differentiation of the germ cell into the spermatozoon through the modification of gene expression. CREM has multiple repressor and activator isoforms whose expression is tissue-cell-type specific and tightly regulated by various factors at the transcriptional, post-transcriptional and post-translational level. The activator isoform CREMτ controls the expression of several relevant genes in post-meiotic stages of spermatogenesis. In addition, exposure to xenobiotics negatively affects CREMτ expression, which is linked to male infertility. On the other hand, antioxidants could have a positive effect on CREMτ expression and improve sperm parameters in idiopathically infertile men. Therefore, CREM expression could be used as a biomarker to detect and even counteract male infertility. This review examines the importance of CREM as a transcription factor for sperm production and its relevance in male fertility, infertility and the response to environmental xenobiotics that may affect CREMτ expression and the downstream regulation that alters male fertility. Also, some health disorders in which CREM expression is altered are discussed.

Neurotoxicity and Oxidative Stress Development in Adult Atya lanipes Shrimp Exposed to Titanium Dioxide Nanoparticles.

Titanium dioxide is a type of nanoparticle that is composed of one titanium atom and two oxygen atoms. One of its physicochemical activities is photolysis, which produces different reactive oxygen species (ROS). Atya lanipes shrimp affect detrital processing and illustrate the potential importance of diversity and nutrient availability to the rest of the food web. It is essential in removing sediments, which have an important role in preventing eutrophication. This study aimed to determine the toxic effect of changes in behavior and levels of oxidative stress due to exposure to titanium dioxide nanoparticles in Atya lanipes and to determine the effective concentration (EC50) for behavioral variables. The concentrations of TiO2 NPs tested were 0.0, 0.50, 1.0, 2.0, and 3.0 mg/L with the positive controls given 100 µg/L of titanium and 3.0 mg/L of TiO2 NPs ± 100 µg/L of titanium. After 24 h of exposure, significant hypoactivity was documented. The EC50 was determined to be a concentration of 0.14 mg/L. After the exposure to 10 mg/L of TiO2 NPs, oxidative stress in gastrointestinal and nervous tissues was documented. The toxic effects of this emerging aquatic pollutant in acute exposure conditions were characterized by sublethal effects such as behavior changes and oxidative stress.

BDNF Influence on Adult Terminal Axon Sprouting after Partial Deafferentation.

BDNF is a neurotrophin family member implicated in many different neuronal functions, from neuronal survival during development to synaptic plasticity associated with processes of learning and memory. Its presence in the oculomotor system has previously been demonstrated, as it regulates afferent composition of extraocular motoneurons and their firing pattern. Moreover, BDNF expression increases after extraocular motoneuron partial deafferentation, in parallel with terminal axon sprouting from the remaining axons. To elucidate whether BDNF could play an active role in this process, we performed partial deafferentation of the medial rectus motoneurons through transection of one of the two main afferents, that is, the ascending tract of Deiters, and injected BDNF into the motoneuron target muscle, the medial rectus. Furthermore, to check whether BDNF could stimulate axon sprouting without lesions, we performed the same experiment without any lesions. Axon terminal sprouting was assessed by calretinin immunostaining, which specifically labels the remaining afferent system on medial rectus motoneurons, the abducens internuclear neurons. The results presented herein show that exogenous BDNF stimulated terminal axon growth, allowing the total recovery of synaptic coverage around the motoneuron somata. Moreover, calretinin staining in the neuropil exceeded that present in the control situation. Thus, BDNF could also stimulate axonal sprouting in the neuropil of intact animals. These results point to an active role of BDNF in plastic adaptations that take place after partial deafferentation.

A Systematic Review of Interventions Based on Judeo-Christian Principles for Patients with Substance Dependence.

Substance use disorders have significant consequences for patients and those around them. A qualitative systematic review was conducted to examine the effectiveness of religiosity and spirituality (R/S) interventions based on Judeo-Christian principles during the multidisciplinary treatment of patients with substance dependence disorder. Studies of patients of both sexes, at any age, of any nationality, and from any geographic location who were participating in treatment programs with religious elements explicitly tied to Christianity or Judaism were eligible for inclusion. Other religious interventions were excluded. Seven electronic databases were searched to identify eligible intervention studies published in English until August 2021. Two reviewers independently screened studies and extracted data. JBI tools were used to assess risk of bias. Of 146 articles retrieved for full text reading, five were ultimately included in this review published from July 2008 to August 2021. The R/S interventions used in the included studies were music therapy in a religious context, reading of the Bible or Torah, reflexive readings, personal prayer and reflection, and religiously integrated cognitive behavioral psychotherapy with or without a comparison group. Despite the broad search, there were found only a small number of studies and little homogeneity in the data of patients that reported positive impacts of treatments. Well-designed controlled studies are needed to truly investigate the efficacy of Judeo-Christian religious interventions for helping people with substance use problems, so as to strengthen the evidence of the benefits of these type of approaches in the treatment of drug addicts.

Combined liver-kidney transplant in polycystic diseases: a case report.

Polycystic liver disease, a hereditary pathology, usually manifests as autosomal dominant polycystic kidney disease. The many cysts in the liver cause massive hepatomegaly, majorly affecting the patient's quality of life. In cases of refractory symptoms, liver transplantation is the only treatment choice. A 43-year-old woman was followed up as a hepatology outpatient in August 2020, with a progressive increase in abdominal volume, lower limb edema, and cachexia. The patient was diagnosed with polycystic renal and liver disease with massive hepatomegaly in March 2021, a combined kidney-liver transplant. Liver size represented 13% of the patient's corporal composition, weighing 8.6kg. The patient was discharged on the 7th postoperative day with no complications. Only 10-20% of patients with polycystic liver disease have clinical manifestations, most of which result from hepatomegaly. An increase in liver volume deteriorates liver function until the condition becomes end-stage liver disease, as kidney function is already compromised; liver-kidney transplantation remains the only treatment choice. The case described drew significant attention to the massive hepatomegaly presented in the patient, with the liver representing over 10% of the patient's body weight, approximately five to six times larger than a normal-sized liver.

Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF.

The potassium chloride cotransporter 2 (KCC2) is the main Cl- extruder in neurons. Any alteration in KCC2 levels leads to changes in Cl- homeostasis and, consequently, in the polarity and amplitude of inhibitory synaptic potentials mediated by GABA or glycine. Axotomy downregulates KCC2 in many different motoneurons and it is suspected that interruption of muscle-derived factors maintaining motoneuron KCC2 expression is in part responsible. In here, we demonstrate that KCC2 is expressed in all oculomotor nuclei of cat and rat, but while trochlear and oculomotor motoneurons downregulate KCC2 after axotomy, expression is unaltered in abducens motoneurons. Exogenous application of vascular endothelial growth factor (VEGF), a neurotrophic factor expressed in muscle, upregulated KCC2 in axotomized abducens motoneurons above control levels. In parallel, a physiological study using cats chronically implanted with electrodes for recording abducens motoneurons in awake animals, demonstrated that inhibitory inputs related to off-fixations and off-directed saccades in VEGF-treated axotomized abducens motoneurons were significantly higher than in control, but eye-related excitatory signals in the on direction were unchanged. This is the first report of lack of KCC2 regulation in a motoneuron type after injury, proposing a role for VEGF in KCC2 regulation and demonstrating the link between KCC2 and synaptic inhibition in awake, behaving animals.

Role of vascular endothelial growth factor as a critical neurotrophic factor for the survival and physiology of motoneurons.

Vascular endothelial growth factor (VEGF) was discovered by its angiogenic activity. However, during evolution, it appeared earlier as a neurotrophic factor required for the development of the nervous system in invertebrates lacking a circulatory system. We aimed at reviewing recent evidence indicating that VEGF has neuroprotective effects in neurons exposed to a variety of insults. Of particular interest is the link established between VEGF and motoneurons, especially after the design of the VEGFδ/δ mutant mice. These mice are characterized by low levels of VEGF and develop muscle weakness and motoneuron degeneration resembling amyotrophic lateral sclerosis. The administration of VEGF through several routes to animal models of amyotrophic lateral sclerosis delays motor impairment and motoneuron degeneration and increases life expectancy. There are new recent advances in the role of VEGF in the physiology of motoneurons. Our experimental aims use the extraocular (abducens) motoneurons lesioned by axotomy as a model for studying VEGF actions. Axotomized abducens motoneurons exhibit severe alterations in their discharge activity and a loss of synaptic boutons. The exogenous administration of VEGF to axotomized abducens motoneurons, either from the transected nerve or intraventricularly, fully restores the synaptic and discharge properties of abducens motoneurons, despite being axotomized. In addition, when an anti-VEGF neutralizing antibody is delivered from the muscle to intact, uninjured abducens motoneurons, these cells display alterations in their discharge pattern and a loss of synaptic boutons that resemble the state of axotomy. All these data indicate that VEGF is an essential neurotrophic factor for motoneurons.

Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein.

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtained only when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

Combined liver-kidney transplant in polycystic diseases: a case report

ABSTRACT Polycystic liver disease, a hereditary pathology, usually manifests as autosomal dominant polycystic kidney disease. The many cysts in the liver cause massive hepatomegaly, majorly affecting the patient's quality of life. In cases of refractory symptoms, liver transplantation is the only treatment choice. A 43-year-old woman was followed up as a hepatology outpatient in August 2020, with a progressive increase in abdominal volume, lower limb edema, and cachexia. The patient was diagnosed with polycystic renal and liver disease with massive hepatomegaly in March 2021, a combined kidney-liver transplant. Liver size represented 13% of the patient's corporal composition, weighing 8.6kg. The patient was discharged on the 7th postoperative day with no complications. Only 10-20% of patients with polycystic liver disease have clinical manifestations, most of which result from hepatomegaly. An increase in liver volume deteriorates liver function until the condition becomes end-stage liver disease, as kidney function is already compromised; liver-kidney transplantation remains the only treatment choice. The case described drew significant attention to the massive hepatomegaly presented in the patient, with the liver representing over 10% of the patient's body weight, approximately five to six times larger than a normal-sized liver.

Eye Movements But Not Vision Drive the Development of Palisade Endings.

Purpose: To test whether visual experience and/or eye movements drive the postnatal development of palisade endings in extraocular muscles. Methods: In three newborn cats, the right eye was covered until 30 days from postnatal (P) day 7 (before opening their eyes), and in three cats both eyes were covered until 45 days, also from P7. To block eye movements, another seven cats received a retrobulbar injection of botulinum neurotoxin A (BoNT-A) into the left orbit at birth and survived for 45 days (three cats) and 95 days (four cats). The distal third of the rectus muscles containing the palisade endings was used for whole-mount preparation and triple-fluorescence labeling with anti-neurofilament along with (1) anti-synaptophysin and phalloidin or (2) anti-growth associated protein 43 (GAP43) and phalloidin. Immunolabeled specimens were analyzed in the confocal laser scanning microscope. Results: After unilateral and bilateral dark rearing, palisade endings were qualitatively and quantitatively equal to those from age-matched controls. After BoNT-A induced eye immobilization for 45 or 95 days, palisade endings were absent in the superior rectus and lateral rectus muscles and only present in the inferior rectus and medial rectus muscle. These BoNT-A-treated palisade endings were rudimentary and reduced in number, and the expression of the neuronal developmental protein GAP43 was significantly reduced. Conclusions: This study demonstrates that eye immobilization, but not visual deprivation, affects palisade ending development. Palisade endings develop in the first month of life, and the present findings indicate that, during this time window, palisade endings are prone to oculomotor perturbations.

Cardiovocal Syndrome - An Aortic Arch Aneurysm As A Rare Cause Of Vocal Cord Paralysis.

INTRODUCTION: Cardiovocal or Ortner's syndrome is a rare cause of vocal cord paralysis. Damage to the left recurrent laryngeal nerve may be caused by an aortic arch aneurysm, in even rarer cases. CLINICAL CASE: A 60-year-old woman presented with hoarseness lasting for six months. Paralysis of the left vocal cord was confirmed with laryngoscopy and an aortic arch aneurysm was diagnosed on chest CT. Despite correction of the aortic aneurysm, her hoarseness did not improve. DISCUSSION: Mediastinal disease may cause vocal cord paralysis, due to the intrathoracic course of the recurrent laryngeal nerve. The assessment of the superior mediastinum on CT is mandatory in these cases. In cardiovocal syndrome, cardiovascular diseases damage the recurrent laryngeal nerve. Aortic aneurysms are a rare cause of Ortner's, especially when they affect the distal portion of the aortic arch and stretch the left recurrent laryngeal nerve at the aortopulmonary window.

VEGF and Neuronal Survival.

Vascular endothelial growth factor (VEGF) is well known for its angiogenic activity, but recent evidence has revealed a neuroprotective action of this factor on injured or diseased neurons. In the present review, we summarize the most relevant findings that have contributed to establish a link between VEGF deficiency and neuronal degeneration. At issue, 1) mutant mice with reduced levels of VEGF show adult-onset muscle weakness and motoneuron degeneration resembling amyotrophic lateral sclerosis (ALS), 2) administration of VEGF to different animal models of motoneuron degeneration improves motor performance and ameliorates motoneuronal degeneration, and 3) there is an association between low plasmatic levels of VEGF and human ALS. Altogether, the results presented in this review highlight VEGF as an essential motoneuron neurotrophic factor endowed with promising therapeutic potential for the treatment of motoneuron disorders.

Giardia duodenalis enolase is secreted as monomer during trophozoite-epithelial cell interactions, activates plasminogen and induces necroptotic damage.

Enolase, a multifunctional protein expressed by multiple pathogens activates plasminogen to promote proteolysis on components of the extracellular matrix, an important event in early host-pathogen interactions. A secreted form of enolase that is released upon the interaction of trophozoites with epithelial cells has been detected in the secretome of G. duodenalis. However, the role of enolase in the host-pathogen interactions remains largely unknown. In this work, the effects of G. duodenalis enolase (Gd-eno) on the epithelial cell model (IEC-6) were analyzed. Firstly, the coding sequence of Giardia enolase was cloned and the recombinant protein used to raise antibodies that were then used to define the localization and role of enolase in epithelial cell-trophozoite interactions. Gd-eno was detected in small cytoplasmic vesicles as well as at the surface and is enriched in the region of the ventral disk of Giardia trophozoites. Moreover, the blocking of the soluble monomeric form of the enzyme, which is secreted upon interaction with IEC-6 cells by the anti-rGd-eno antibodies, significantly inhibited trophozoite attachment to intestinal IEC-6 cell monolayers. Further, rGd-eno was able to bind human plasminogen (HsPlg) and enhanced plasmin activity in vitro when the trophozoites were incubated with the intrinsic plasminogen activators of epithelial cells. In IEC-6 cells, rGd-eno treatment induced a profuse cell damage characterized by copious vacuolization, intercellular separation and detachment from the substrate; this effect was inhibited by either anti-Gd-eno Abs or the plasmin inhibitor ϵ- aminocaproic acid. Lastly, we established that in epithelial cells rGd-eno treatment induced a necroptotic-like process mediated by tumor necrosis factor α (TNF-α) and the apoptosis inducing factor (AIF), but independent of caspase-3. All together, these results suggest that Giardia enolase is a secreted moonlighting protein that stimulates a necroptotic-like process in IEC-6 epithelial cells via plasminogen activation along to TNFα and AIF activities and must be considered as a virulence factor.

Extraocular Motoneurons and Neurotrophism.

Extraocular motoneurons are located in three brainstem nuclei: the abducens, trochlear and oculomotor. They control all types of eye movements by innervating three pairs of agonistic/antagonistic extraocular muscles. They exhibit a tonic-phasic discharge pattern, demonstrating sensitivity to eye position and sensitivity to eye velocity. According to their innervation pattern, extraocular muscle fibers can be classified as singly innervated muscle fiber (SIF), or the peculiar multiply innervated muscle fiber (MIF). SIF motoneurons show anatomical and physiological differences with MIF motoneurons. The latter are smaller and display lower eye position and velocity sensitivities as compared with SIF motoneurons.

Giardia duodenalis carries out canonical homologous recombination and single-strand annealing.

In the past decades, the ability of Giardia duodenalis to perform homologous recombination has been suggested, supported by the observations of genomic integration of foreign plasmids and the disruption of genes using CRISPR technology. Unfortunately, the direct study of a HR mechanism has not been addressed, which would be pertinent in a minimalist organism lacking fundamental DNA-repair elements and even complete pathways. In addition, the constant ploidy changes through the life cycle of this parasite highlight the conservation and relevance of homologous recombination in maintaining genomic stability. In this research, we analyzed different recombinable plasmid systems and their outcomes after G. duodenalis transfection, using this approach we determined genomic, intra-plasmid and inter-plasmid recombination, moreover, we examined the presence of the non-conservative single-strand annealing pathway. With the intention of corroborating that the observed processes were done by homologous recombination, we used a chemical inhibitor named Mirin, which specifically inhibits Mre11 3'- 5' exonuclease activity, one of the first steps involved in homologous recombination and fundamental to success in repairing. Overall, these results describe the multiple recombinational substrates used by G. duodenalis to achieve HR and demonstrate the presence and use of single-strand annealing recombination.

Cancer in Amphibia, a rare phenomenon?

Compared to other animals, the spontaneous occurrence of tumors in wild amphibians is relatively rare, generally limited to specific populations or species. The number of reports of spontaneous tumors in amphibians known up to 1986 was 491 cases in anurans and about 253 cases in urodeles. Similarly, there have been many, unsuccessful attempts to chemically or biologically induce tumors in amphibians. With these considerations, it is inevitable to wonder: do urodeles and anurans have an inherent resistance to cancer? Here, we review the spontaneous and induced occurrence of tumors in amphibians in a timeline, as well as failed attempts to induce tumors in these amphibians. Indeed, recent studies seem to indicate that there is a relationship between regeneration and cancer because regenerating tissues seem to resist tumorigenesis, as opposed to nonregenerative tissues of the same amphibian models. Although the mechanisms that allow regenerating tissues to resist tumorigenesis have not been elucidated, it is worth to note that, in addition to the apparent relationship between regeneration and cancer, amphibians possess characteristics that could contribute to their ability to resist the development of neoplastic events. The implications of these features in cancer susceptibility are discussed.

Implementation of a tunable t-CRISPRi system for gene regulation in Giardia duodenalis.

Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.