Dispersal history of SARS-CoV-2 in Galicia, Spain.

The dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron-BA.1 variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation.

The differentiation of self-renewing progenitor cells requires not only the regulation of lineage- and developmental stage-specific genes but also the coordinated adaptation of housekeeping functions from a metabolically active, proliferative state toward quiescence. How metabolic and cell-cycle states are coordinated with the regulation of cell type-specific genes is an important question, because dissociation between differentiation, cell cycle, and metabolic states is a hallmark of cancer. Here, we use a model system to systematically identify key transcriptional regulators of Ikaros-dependent B cell-progenitor differentiation. We find that the coordinated regulation of housekeeping functions and tissue-specific gene expression requires a feedforward circuit whereby Ikaros down-regulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping states can be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.

The Sponge-Derived Brominated Compound Aeroplysinin-1 Impairs the Endothelial Inflammatory Response through Inhibition of the NF-κB Pathway.

(+)-Aeroplysinin-1 (Apl-1) is a brominated compound isolated from the marine sponge Aplysina aerophoba that exhibits pleiotropic bioactive effects, impairing cell growth in cancer cells, inhibiting angiogenesis in vitro and in vivo and modulating the redox status of different cell types, among other reported activities. In addition to the aforementioned effects, the anti-inflammatory potential of this natural compound was explored in previous work of our laboratory, but the mechanism of action underlying this effect was not described. In this work, we delve into the anti-inflammatory effect of Apl-1 in the context of vascular endothelial cells in vitro, providing new data regarding the molecular mechanism underlying this activity. The characterization of the mechanism of action points to an inhibitory effect of Apl-1 on the NF-κB pathway, one of the main axes involved in endothelial response during inflammatory events. Our results show that Apl-1 can inhibit the expression of pro-inflammatory genes in tumor necrosis factor alpha (TNF-α)- and lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), targeting the nuclear factor kappa B subunit (NF-κB) pathway through a mechanism of action involving the inhibition of I kappa B kinase (IKK) complex phosphorylation and RelA/p65 nuclear import. In addition, Apl-1 prevented the phosphorylation of Akt induced by TNF-α in HUVECs, probably supporting the inhibitory effect of this compound in the NF-κB pathway. Experimental evidence reported in this work opens the door to the potential pharmacological use of this compound as an anti-inflammatory agent in diseases that course with a pathological endothelial response to inflammation, such as atherosclerosis.

The Immunomodulator Dimethyl Itaconate Inhibits Several Key Steps of Angiogenesis in Cultured Endothelial Cells.

The dimethyl derivative of the immunomodulator itaconate has been previously shown to have anti-inflammatory, anti-oxidative, and immunomodulatory effects. In the present work, we evaluate the potential of dimethyl itaconate as an anti-angiogenic compound by using cultured endothelial cells and several in vitro assays that simulate key steps of the angiogenic process, including endothelial cell proliferation, migration, invasion, and tube formation. Our results show that dimethyl itaconate interferes with all the previously mentioned steps of the angiogenic process, suggesting that dimethyl itaconate behaves as an anti-angiogenic compound.

Localization of serum resistance-associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei.

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.

Genome-wide identification of Ikaros targets elucidates its contribution to mouse B-cell lineage specification and pre-B-cell differentiation.

Ikaros family DNA-binding proteins are critical regulators of B-cell development. Because the current knowledge of Ikaros targets in B-cell progenitors is limited, we have identified genes that are bound and regulated by Ikaros in pre-B cells. To elucidate the role of Ikaros in B-cell lineage specification and differentiation, we analyzed the differential expression of Ikaros targets during the progression of multipotent to lymphoid-restricted progenitors, B- and T-cell lineage specification, and progression along the B-cell lineage. Ikaros targets accounted for one-half of all genes up-regulated during B-cell lineage specification in vivo, explaining the essential role of Ikaros in this process. Expression of the Ikaros paralogs Ikzf1 and Ikzf3 increases incrementally during B-cell progenitor differentiation, and, remarkably, inducible Ikaros expression in cycling pre-B cells was sufficient to drive transcriptional changes resembling the differentiation of cycling to resting pre-Bcells in vivo. The data suggest that Ikaros transcription factor dosage drives the progression of progenitors along a predetermined lineage by regulating multiple targets in key pathways, including pre-B­cell receptor signaling, cell cycle progression, and lymphocyte receptor rearrangement.Our approachmay be of general use to map the contribution of transcription factors to cell lineage commitment and differentiation.

Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei.

African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms, the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases, TbTOR1 and TbTOR2, and two TOR complexes, TbTORC1 and TbTORC2. Surprisingly, two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor), a major vault protein, and LST8 to form a unique TOR complex, TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.

Pearls & Oy-sters: A Challenging Optic Neuropathy-What Not to Miss in Optic Nerve Sheath Enhancement.

Optic neuropathies include a wide range of disorders from ischemic, toxic, demyelinating, or inflammatory processes with acute/subacute onset to more gradual compressive or genetic etiologies. Accurate clinical history and multimodality optic nerve imaging including MRI and optical coherence tomography have greatly improved the diagnosis of patients with optic neuropathies. We report a case of a woman with severe monocular visual acuity deficit. Optic nerve sheath enhancement seen on MRI led to a broad differential diagnosis including demyelinating causes, optic nerve sheath meningioma (ONSM), tuberculosis, and sarcoid optic neuropathy. Lack of response to treatment with steroids or plasmapheresis led to biopsy, which confirmed the diagnosis of ONSM.

Dispersal history of SARS-CoV-2 in Galicia, Spain.

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Anti-angiogenic effects of oleacein and oleocanthal: New bioactivities of compounds from Extra Virgin Olive Oil.

Phenolic compounds play a key role in the health benefits of Extra Virgin Olive Oil (EVOO). Among these molecules, the focus has been recently put on (-)-oleocanthal and (-)-oleacein, for which anti-cancer and angiogenesis-related findings have been reported. Here, we explored the modulatory action of (-)-oleocanthal and (-)-oleacein on angiogenesis, the process by which new vessels are created from pre-existent ones, which is directly linked to tumor progression and other pathological conditions. Two in vivo models strongly sustained by angiogenesis, and an in vitro model of endothelial cells to study different steps of angiogenesis, were used. In vivo evidence pointed to the anti-angiogenic effects of both compounds in vivo. In vitro, (-)-oleacein and (-)-oleocanthal inhibited the proliferation, invasion, and tube formation of endothelial cells, and (-)-oleacein significantly repressed migration and induced apoptosis in these cells. Mechanistically, the compounds modulated signaling pathways related to survival and proliferation, all at concentrations of physiological relevance for humans. We propose (-)-oleacein and (-)-oleocanthal as good candidates for angioprevention and for further studies as modulators of angiogenesis in clinical interventions, and as interesting functional claims for the food industry. Chemical compounds studied in this article: Oleocanthal (PubChem CID: 11652416); Oleacein (PubChem CID: 18684078).

Danthron, an Anthraquinone Isolated from a Marine Fungus, Is a New Inhibitor of Angiogenesis Exhibiting Interesting Antitumor and Antioxidant Properties.

The role played by a sustained angiogenesis in cancer and other diseases stimulates the interest in the search for new antiangiogenic drugs. In this manuscript, we provide evidence that 1,8- dihydroxy-9,10-anthraquinone (danthron), isolated from the fermentation broth of the marine fungus Chromolaenicola sp. (HL-114-33-R04), is a new inhibitor of angiogenesis. The results obtained with the in vivo CAM assay indicate that danthron is a potent antiangiogenic compound. In vitro studies with human umbilical endothelial cells (HUVEC) reveal that this anthraquinone inhibits certain key functions of activated endothelial cells, including proliferation, proteolytic and invasive capabilities and tube formation. In vitro studies with human breast carcinoma MDA-MB231 and fibrosarcoma HT1080 cell lines suggest a moderate antitumor and antimetastatic activity of this compound. Antioxidant properties of danthron are evidenced by the observation that it reduces the intracellular reactive oxygen species production and increases the amount of intracellular sulfhydryl groups in endothelial and tumor cells. These results support a putative role of danthron as a new antiangiogenic drug with potential application in the treatment and angioprevention of cancer and other angiogenesis-dependent diseases.

Clinicopathological Profiles Associated with Discordant RAS Mutational Status between Liquid and Tissue Biopsies in a Real-World Cohort of Metastatic Colorectal Cancer.

We aimed to identify common mCRC profiles associated with a discordant mutational status of RAS between the standard of care (SoC) tumour tissue tests and ctDNA tests to understand ctDNA detection and improve treatment responses. This was a multicentre, retrospective and prospective study. A total of 366 Spanish mCRC patients were independently recruited. BEAMing ddPCR technology was employed to detect ctDNA RAS mutations, and logistic regression analyses were performed to investigate clinicopathological factors associated with discordance. The highest concordance ratios were observed in profiles with multiple metastatic sites when the liver was present (89.7%; 95% CI 84.8-93.2), profiles with synchronous disease without primary tumour resection (90.2%; 95% CI 83.6-94.3) and profiles with mCRC originating in the left colon (91.3%; 95% CI 85.0-95.0). Metachronous disease originating in the right colon (OR = 6.1; 95% CI 1.7-26.5; p-value = 0.006) or rectum (OR = 5.0; 95% CI 1.5-17.8; p-value = 0.009) showed the highest probability of discrepancies. Primary tumour resection and a higher frequency of single metastases in the peritoneum or lungs in these patients were associated with reduced plasmatic mutation allele fractions (MAFs) and an increased probability of showing false-negative genotypes. Additional testing of patients with mCRC originating in the right colon or rectum with a single non-mutated ctDNA test is advised before the choice of therapy.

Effects on venous flow of transcutaneous electrical stimulation, neuromuscular stimulation, and sham stimulation on soleus muscle: A randomized crossover study in healthy subjects.

BACKGROUND: Activation of venous flow has been shown with different types of electrical stimulation. The aim of this study is to compare the hemodynamic effects of transcutaneous electrical nerve stimulation (TENS), neuromuscular electrical stimulation (NMES), and sham stimulation on healthy young people. METHODS: This randomized crossover study was conducted during June 2018 in the Faculty of Physical Therapy of A Coruña (Spain). Twenty-four university students (50% male) received in a randomized order 5 Hz-TENS, NMES, and sham stimulation on soleus muscle. Flow volume (FV) and peak velocity (PV) from popliteal vein were recorded via Doppler ultrasound, and relative changes from baseline were determined. Discomfort among the 3 stimulations was also compared. RESULTS: The differences among the 3 stimulations were assessed using the ANOVA for repeated measured, the Friedman test and the Kendall tau test, according to the type of measurement to be compared. FV (mL/min) and PV (cm/s) increased significantly after NMES (percentual increase 37.2 ± 62.0%, P = .002; 264.4 ± 152.2%, P < .001, respectively) and TENS (226.2 ± 190.3%, P < .001; 202.7 ± 144.6%, P < .001, respectively). These percentual changes from basal level in hemodynamics were statistically different to those after placebo, which was ineffective enhancing hemodynamics. The improvements in FV were statistically higher with TENS than with NMES (P < .001), but there was no statistical difference in PV (P = .531). Despite NMES was applied at a significantly lower amplitude than TENS (P < .001), NMES protocol was the worst tolerated, though the differences in discomfort were not statistically significant. CONCLUSION: Both active electrical protocols but not sham stimulation increased hemodynamics in healthy people. TENS obtained higher flow volume increase from baseline than NMES, considered globally at not only in its on-time.

RNA pol II subunit RPB7 is required for RNA pol I-mediated transcription in Trypanosoma brucei.

In the protozoan parasite Trypanosoma brucei, the two main surface glycoprotein genes are transcribed by RNA polymerase I (pol I) instead of RNA pol II, the polymerase committed to the production of mRNA in eukaryotes. This unusual feature might be accomplished by the recruitment of specific subunits or cofactors that allow pol I to transcribe protein-coding RNAs. Here, we report that transcription mediated by pol I requires TbRPB7, a dissociable subunit of the pol II complex. TbRPB7 was found to interact with two pol I-specific subunits, TbRPA1 and TbRPB6z. Pol I-specific transcription was affected on depletion of TbRPB7 in run-on assays, whereas recombinant TbRPB7 increased transcription driven by a pol I promoter. These results represent a unique example of a functional RNA polymerase chimaera consisting of a core pol I complex that recruits a specific pol II subunit.

[Prevalence and determinants of breastfeeding: the Zorrotzaurre study]. / Prevalencia y determinantes de la lactancia materna: estudio Zorrotzaurre.

INTRODUCTION: Introduction and objectives: breastfeeding (BF) is a feeding method that provides multiple benefits for the health of infants and their mothers. This study aimed to determine the prevalence of BF during the first year of life of children of women who gave birth in a private clinic in Biscay, Basque Country, Spain, and to identify the facilitating determinants and reasons for abandonment. Method: an observational, descriptive, longitudinal and prospective study in a random sample of 453 newborns (NBs) recruited between 2016 and 2017. Results: in all, 366 women agreed to participate in all the study phases. The prevalence of exclusive breastfeeding (EBF) was 51.7 % at baseline, 77.1 % at discharge, and 21.6 % after sixth months; and that of BF, 87.1 % at the beginning, 48.4 % at month six, and 20.6 % at one year. The facilitating factors of EBF were: at the beginning, not using a nest or breast pump; 15 days of satisfaction with LM and not using a pacifier or breast pump; 4 months of satisfaction with LM; 6 months attending Lactation Support Groups (GAL) and not introducing complementary feeding (CA); and those of LM at 1 year, attending GAL. The main reasons for abandonment were: own initiative, incorporation to work, and little weight gain by the NB. Conclusions: one in 5 newborns received EBF up to 6 months and BF up to one year. It would be necessary to promote strategies that favor breastfeeding, such as eliminating the nest, advising against breast pumps and pacifiers at the beginning, starting CA from the sixth month, and organizing GALs during the first year.

INTRODUCCIÓN: Introducción y objetivos: la lactancia materna (LM) es un método de alimentación infantil que aporta múltiples beneficios para la salud de los lactantes y las madres. Este estudio pretende determinar la prevalencia de la LM durante el primer año de vida de los hijos/as de una serie de mujeres que dan a luz en una clínica privada de Bizkaia, e identificar los determinantes facilitadores y los motivos de abandono. Método: estudio observacional, descriptivo, longitudinal y prospectivo de una muestra aleatoria de 453 recién nacidos (RN), reclutada entre 2016 y 2017. Resultados: en total, 366 mujeres aceptaron participar en todas las fases de estudio. La prevalencia de la lactancia materna exclusiva (LME) fue del 51,7 % al inicio, del 77,1 % al alta y del 21,6 % al sexto mes; y la de la LM, del 87,1 % al inicio, del 48,4 % al sexto mes y del 20,6 % al año. Los factores facilitadores de la LME fueron: al inicio, no utilizar nido ni sacaleches; 15 días de satisfacción con la LM y no utilizar chupete ni sacaleches; 4 meses de satisfacción con la LM; 6 meses de acudir a Grupos de Apoyo a la Lactancia (GAL) y no introducir alimentación complementaria (AC); los de la LM al año, acudir a GAL. Los principales motivos de abandono fueron: la iniciativa propia, la incorporación al trabajo y la escasa ganancia de peso del RN. Conclusiones: uno de cada 5 RN recibieron LME hasta los 6 meses y LM hasta el año. Sería necesario promover estrategias que favorezcan el amamantamiento, como: suprimir el nido, desaconsejar el sacaleches y el chupete al inicio, iniciar la AC a partir del sexto mes y organizar GAL durante el primer año.

Evaluation of a Targeted Next-Generation Sequencing Panel for the Non-Invasive Detection of Variants in Circulating DNA of Colorectal Cancer.

Molecular profiling of circulating cell-free DNA (cfDNA) has shown utility for the management of colorectal cancer (CRC). TruSight Tumor 170 (TST170) is a next-generation sequencing (NGS) panel that covers 170 cancer-related genes, including KRAS, which is a key driver gene in CRC. We evaluated the capacity of TST170 to detect gene variants in cfDNA from a retrospective cohort of 20 metastatic CRC patients with known KRAS variants in tumor tissue and in cfDNA previously analyzed by pyrosequencing and BEAMing, respectively. The cfDNA of most of the patients (95%) was successfully sequenced. We frequently detected variants with clinical significance in KRAS (79%, 15/19) and PIK3CA (26%, 5/19) genes. Variants with potential clinical significance were also identified in another 27 cancer genes, such as APC. The type of KRAS variant detected in cfDNA by TST170 showed high concordance with those detected in tumor tissue (77%), and very high concordance with cfDNA analyzed by BEAMing (94%). The variant allele fractions for KRAS obtained in cfDNA by TST170 and BEAMing correlated strongly. This proof-of-principle study indicates that targeted NGS analysis of cfDNA with TST170 could be useful for non-invasive detection of gene variants in metastatic CRC patients, providing an assay that could be easily implemented for detecting somatic alterations in the clinic.

Association between general and central adiposity and development of hypertension in early childhood.

OBJECTIVES: To evaluate the association of general and abdominal obesity with high blood pressure in young children. METHODS: A longitudinal study including 1796 participants from the Madrid region (Spain) with baseline at age 4 years and a follow-up 2 years later. Blood pressure, body mass index and waist circumference were measured during a physical examination. We evaluated the association between obesity at baseline and weight changes between the ages of 4 and 6 years and high blood pressure. Data were analysed using linear and logistic regressions adjusted for covariates. RESULTS: Obese 4 year olds (general or abdominal obesity) experienced an average 4-5 mmHg increase in systolic blood pressure and a 2.5-3 mmHg increase in diastolic blood pressure by the age of 6 years. Compared to children maintaining a non-excess weight (based on body mass index) during follow-up incident and persistent cases of excess weight (overweight or obesity) had an odds ratio (OR) for high blood pressure of 2.49 (95% confidence interval (CI) 1.50-4.13) and OR 2.54 (95% CI 1.27-5.07), respectively. Regarding abdominal obesity we estimated OR 2.81 (95% CI 0.98-8.02) for incident cases and OR 3.42 (95% CI 1.38-8.49) for persistent cases. Similar estimates for the waist-height ratio were observed. Individuals who experienced remission to non-excess weight did not have an increased risk of high blood pressure. CONCLUSIONS: We observed an increased risk for high blood pressure among 4-year-olds who presented with persistent or incident cases of excess weight (body mass index) or abdominal obesity after 2 years of follow-up. Children with excess weight or obesity at baseline who remitted to non-excess weight did not exhibit an increased risk of high blood pressure.

STATegra, a comprehensive multi-omics dataset of B-cell differentiation in mouse.

Multi-omics approaches use a diversity of high-throughput technologies to profile the different molecular layers of living cells. Ideally, the integration of this information should result in comprehensive systems models of cellular physiology and regulation. However, most multi-omics projects still include a limited number of molecular assays and there have been very few multi-omic studies that evaluate dynamic processes such as cellular growth, development and adaptation. Hence, we lack formal analysis methods and comprehensive multi-omics datasets that can be leveraged to develop true multi-layered models for dynamic cellular systems. Here we present the STATegra multi-omics dataset that combines measurements from up to 10 different omics technologies applied to the same biological system, namely the well-studied mouse pre-B-cell differentiation. STATegra includes high-throughput measurements of chromatin structure, gene expression, proteomics and metabolomics, and it is complemented with single-cell data. To our knowledge, the STATegra collection is the most diverse multi-omics dataset describing a dynamic biological system.

Effects of transcutaneous electrical nerve stimulation via peroneal nerve or soleus muscle on venous flow: A randomized cross-over study in healthy subjects.

BACKGROUND: Transcutaneous electrical nerve stimulation (TENS) is used to prevent venous stasis and thromboembolism. However, best electrostimulation parameters have yet to be established. The aim of the study was to compare the hemodynamic effects and the participants' relative discomfort of 3 TENS sequences at the maximum tolerated intensity stimulus. METHODS: Twenty-four healthy university students (50% male) participated in a cross-over, randomized study. Each participant received 2 TENS sequences on peroneal nerve at 1 and 5 Hz, and the third one on soleus muscle at 5 Hz. Popliteal flow volume (FV) and peak velocity (PV) were measured using Doppler ultrasound and the relative change from basal values was recorded. Discomfort questionnaires -visual analogue scale (VAS) and verbal rating scale (VRS)- were also administered to compare sensations among the three applications. RESULTS: All interventions produced significant hemodynamic responses compared to baseline. Both 5 Hz applications obtained higher FV increments than 1 Hz TENS (P < .001). The muscle application resulted in the lowest PV increment (P < .001). TENS at 5 Hz on nerve location was the worst tolerated, with higher values in VRS (P = .056) and VAS (P = .11), although not significant. CONCLUSION: TENS at 5 Hz on soleus site may be the most appropriate protocol for enhancing venous return.