Advancing functional connectivity research from association to causation. Reid AT, Headley DB, Mill RD, Sanchez-Romero R, Uddin LQ, Marinazzo D, et al. Nat Neurosci. 2019 Oct 14. DOI: 10.1038/s41593-019-0510-4. [Epub ahead of print]

Cognition and behavior emerge from brain network interactions, such that investigating causal interactions should be central to the study of brain function. Approaches that characterize statistical associations among neural time series-functional connectivity (FC) methods-are likely a good starting point for estimating brain network interactions. Yet only a subset of FC methods (‘effective connectivity’) is explicitly designed to infer causal interactions from statistical associations. Here we incorporate best practices from diverse areas of FC research to illustrate how FC methods can be refined to improve inferences about neural mechanisms, with properties of causal neural interactions as a common ontology to facilitate cumulative progress across FC approaches. We further demonstrate how the most common FC measures (correlation and coherence) reduce the set of likely causal models, facilitating causal inferences despite major limitations. Alternative FC measures are suggested to immediately start improving causal inferences beyond these common FC measures.

Age at start of using tobacco on the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium (INHANCE). Chang CP, Chang SC, Chuang SC, Berthiller J, Ferro G, Matsuo K, et al. Cancer Epidemiol. 2019 Oct 3;63:101615. DOI: 10.1016/j.canep.2019.101615. [Epub ahead of print]

Background Tobacco use is a well-established risk factor for head and neck cancer (HNC). However, less is known about the potential impact of exposure to tobacco at an early age on HNC risk. Methods We analyzed individual-level data on ever tobacco smokers from 27 case-control studies (17,146 HNC cases and 17,449 controls) in the International Head and Neck Cancer Epidemiology (INHANCE) consortium. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using random-effects logistic regression models. Results Without adjusting for tobacco packyears, we observed that younger age at starting tobacco use was associated with an increased HNC risk for ever smokers (OR_{<10 years vs. ≥30 years}: 1.64, 95% CI: 1.35, 1.97). However, the observed association between age at starting tobacco use and HNC risk became null after adjusting for tobacco packyears (OR_{<10 years vs. ≥30 years}: 0.97, 95% CI: 0.80, 1.19). In the stratified analyses on HNC subsites by tobacco packyears or years since quitting, no difference in the association between age at start and HNC risk was observed. Conclusions Results from this pooled analysis suggest that increased HNC risks observed with earlier age at starting tobacco smoking are largely due to longer duration and higher cumulative tobacco exposures.

 Analysis of potential anti-aging beverage Pru, a traditional Cuban refreshment, by desorption electrospray ionization-mass spectrometry and FTICR tandem mass spectrometry. Mamun MA, Gonzalez TV, Islam A, Sato T, Sato S, Ito TK, et al. J Food Drug Anal. 2019 Oct;27(4):833–840.

Aging has been established as a major risk factor for prevalent diseases and hence, the development of anti-aging medicines is of great importance. Recently, herbal fermented beverages have emerged as a promising source of potential anti-aging drug. Pru, a traditional Cuban refreshment produced by decoction and fermentation of multispecies plants with sugar, has been consumed for many years and is claimed to have multiple medicinal properties. Besides the traditional method, Pru is also manufactured industrially. The present study analyzed the major components of both traditional Pru (TP) and industrial Pru (IP) to reveal their potential application in promoting the health span. We performed desorption electrospray ionization-mass spectrometry (DESI-MS) and acquired mass spectra by scanning over the 50-1200 m/z range in both positive and negative ion modes. Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry (MS/MS) was performed for validating the compound assignments. Three important compounds were identified by comparing the MS and MS/MS spectra with reported literature and the online database. One of the identified compounds, gluconic acid, was found to be the most abundant shared metabolite between TP and IP whereas the other two compounds, magnoflorine and levan were exclusively detected in TP. The present study is the first report of component profiling in Cuban traditional and industrial Pru using DESI-MS and FTICR MS/MS, and reveals the potential application of Pru as a health-promoting agent.

 Anatomical connections underlying personally-familiar face processing. Góngora D, Castro-Laguardia AM, Pérez J, Valdés-Sosa P, Bobes MA. PLoS One. 2019 Sep 11;14(9):e0222087. doi: 10.1371/journal.pone.0222087. eCollection 2019.

Familiar face processing involves face specific regions (the core face system) as well as other non-specific areas related to processing of person-related information (the extended face system). The connections between core and extended face system areas must be critical for face recognition. Some studies have explored the connectivity pattern of unfamiliar face responding area, but none have explored those areas related to face familiarity processing in the extended system. To study these connections, diffusion weighted imaging with probabilistic tractography was used to estimate the white-matter pathways between core and extended system regions, which were defined from functional magnetic resonance imaging responses to personally-familiar faces. Strong white matter connections were found between occipitotemporal face areas (OFA/FFA) with superior temporal sulcus and insula suggesting the possible existence of direct anatomical connections from face-specific areas to frontal nodes that could underlay the processing of emotional information associated to familiar faces.

 Antibiotics for asymptomatic bacteriuria in pregnancy. Smaill FM, Vazquez JC. Cochrane Database Syst Rev. 2019 Nov 25;2019(11). DOI: 10.1002/14651858.CD000490.pub4.

Background Asymptomatic bacteriuria is a bacterial infection of the urine without any of the typical symptoms that are associated with a urinary infection, and occurs in 2% to 15% of pregnancies. If left untreated, up to 30% of mothers will develop acute pyelonephritis. Asymptomatic bacteriuria has been associated with low birthweight and preterm birth. This is an update of a review last published in 2015. Objectives To assess the effect of antibiotic treatment for asymptomatic bacteriuria on the development of pyelonephritis and the risk of low birthweight and preterm birth. Search methods For this update, we searched the Cochrane Pregnancy and Childbirth’s Trials Register, ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform (ICTRP) on 4 November 2018, and reference lists of retrieved studies. Selection criteria Randomised controlled trials (RCT) comparing antibiotic treatment with placebo or no treatment in pregnant women with asymptomatic bacteriuria found on antenatal screening. Trials using a cluster-RCT design and quasi-RCTs were eligible for inclusion, as were trials published in abstract or letter form, but cross-over studies were not. Data collection and analysis Two review authors independently assessed trials for inclusion and risk of bias, extracted data, and checked for accuracy. We assessed the quality of the evidence using the GRADE approach. Main results We included 15 studies, involving over 2000 women. Antibiotic treatment compared with placebo or no treatment may reduce the incidence of pyelonephritis (average risk ratio (RR) 0.24, 95% confidence interval (CI) 0.13 to 0.41; 12 studies, 2017 women; low-certainty evidence). Antibiotic treatment may be associated with a reduction in the incidence of preterm birth (RR 0.34, 95% CI 0.13 to 0.88; 3 studies, 327 women; low-certainty evidence), and low birthweight babies (average RR 0.64, 95% CI 0.45 to 0.93; 6 studies, 1437 babies; low-certainty evidence). There may be a reduction in persistent bacteriuria at the time of delivery (average RR 0.30, 95% CI 0.18 to 0.53; 4 studies; 596 women), but the results were inconclusive for serious adverse neonatal outcomes (average RR 0.64, 95% CI 0.23 to 1.79, 3 studies; 549 babies). There were very limited data on which to estimate the effect of antibiotics on other infant outcomes, and maternal adverse effects were rarely described. Overall, we judged only one trial at low risk of bias across all domains; the other 14 studies were assessed as high or unclear risk of bias. Many studies lacked an adequate description of methods, and we could only judge the risk of bias as unclear, but in most studies, we assessed at least one domain at high risk of bias. We assessed the quality of the evidence for the three primary outcomes with GRADE software, and found low-certainty evidence for pyelonephritis, preterm birth, and birthweight less than 2500 g. Author’s conclusions Antibiotic treatment may be effective in reducing the risk of pyelonephritis in pregnancy, but our confidence in the effect estimate is limited given the low certainty of the evidence. There may be a reduction in preterm birth and low birthweight with antibiotic treatment, consistent with theories about the role of infection in adverse pregnancy outcomes, but again, the confidence in the effect is limited given the low certainty of the evidence. Research implications identified in this review include the need for an up-to-date cost-effectiveness evaluation of diagnostic algorithms, and more evidence to learn whether there is a low-risk group of women who are unlikely to benefit from treatment of asymptomatic bacteriuria.

Bixa orellana L. (Bixaceae) and Dysphania ambrosioides (L.) Mosyakin & Clemants (Amaranthaceae) Essential Oils Formulated in Nanocochleates against Leishmania amazonensis. Machín L, Tamargo B, Piñón A, Atíes RC, Scull R, Setzer WN, et al. Molecules. 2019 Nov 20;24(23). pii: E4222. DOI: 10.3390/molecules24234222.

Leishmaniasis is a group of neglected tropical diseases caused by protozoan parasites of the Leishmania genus. The absence of effective vaccines and the limitations of current treatments make the search for effective therapies a real need. Different plant-derived essential oils (EOs) have shown antileishmanial effects, in particular from Bixa orellana L. (EO-Bo) and Dysphania ambrosioides (L.) Mosyakin & Clemants (EO-Da). In the present study, the EO-Bo and EO-Da, formulated in nanocochleates (EO-Bo-NC and EO-Da-NC, respectively), were evaluated in vitro and in vivo against L. amazonensis. The EO-Bo-NC and EO-Da-NC did not increase the in vitro inhibitory activity of the EOs, although the EO-Bo-NC showed reduced cytotoxic effects. In the animal model, both formulations (30 mg/kg/intralesional route/every 4 days/4 times) showed no deaths or weight loss greater than 10%. In the animal (mouse) model, EO-Bo-NC contributed to the control of infection (p < 0.05) in comparison with EO-Bo treatment, while the mice treated with EO-Da-NC exhibited larger lesions (p < 0.05) compared to those treated with EO-Da. The enhanced in vivo activity observed for EO-Bo-NC suggests that lipid-based nanoformulations like nanocochleates should be explored for their potential in the proper delivery of drugs, and in particular, the delivery of hydrophobic materials for effective cutaneous leishmaniasis treatment.

 Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity. Vicente FEM, González-García M, Díaz Pico E, Moreno-Castillo E, Garay HE, Rosi PE, et al. ACS Omega. 2019 Nov 5;4(21):19081–5.

Following the information obtained by a rational design study, a cyclic and helical-stabilized analogue of the peptide Cm-p5 was synthetized. The cyclic monomer showed an increased activity in vitro against Candida albicans and Candida parapsilosis, compared to Cm-p5. Initially, 14 mutants of Cm-p5 were synthesized following a rational design to improve the antifungal activity and pharmacological properties. Antimicrobial testing showed that the activity was lost in each of these 14 analogues, suggesting, as a main conclusion, that a Glu-His salt bridge could stabilize Cm-p5 helical conformation during the interaction with the plasma membrane. A derivative, obtained by substitution of Glu and His for Cys, was synthesized and oxidized with the generation of a cyclic monomer with improved antifungal activity. In addition, two dimers were generated during the oxidation procedure, a parallel and antiparallel one. The dimers showed a helical secondary structure in water, whereas the cyclic monomer only showed this conformation in SDS. Molecular dynamic simulations confirmed the helical stabilizations for all of them, therefore indicating the possible essential role of the Glu-His salt bridge. In addition, the antiparallel dimer showed a moderate activity against Pseudomonas aeruginosa and a significant activity against Listeria monocytogenes. Neither the cyclic monomer nor the dimers were toxic against macrophages or THP-1 human cells. Due to its increased capacity for fungal control compared to fluconazole, its low cytotoxicity, together with a stabilized α-helix and disulfide bridges, that may advance its metabolic stability, and in vivo activity, the new cyclic Cm-p5 monomer represents a potential systemic antifungal therapeutic candidate.

 Estimating Exposome Score for Schizophrenia Using Predictive Modeling Approach in Two Independent Samples: The Results From the EUGEI Study. Pries LK, Lage-Castellanos A, Delespaul P, Kenis G, Luykx JJ, Lin BD, et al. Schizophr Bull. 2019 Sep 11;45(5):960–5.

Exposures constitute a dense network of the environment: exposome. Here, we argue for embracing the exposome paradigm to investigate the sum of nongenetic “risk” and show how predictive modeling approaches can be used to construct an exposome score (ES; an aggregated score of exposures) for schizophrenia. The training dataset consisted of patients with schizophrenia and controls, whereas the independent validation dataset consisted of patients, their unaffected siblings, and controls. Binary exposures were cannabis use, hearing impairment, winter birth, bullying, and emotional, physical, and sexual abuse along with physical and emotional neglect. We applied logistic regression (LR), Gaussian Naive Bayes (GNB), the least absolute shrinkage and selection operator (LASSO), and Ridge penalized classification models to the training dataset. ESs, the sum of weighted exposures based on coefficients from each model, were calculated in the validation dataset. In addition, we estimated ES based on meta-analyses and a simple sum score of exposures. Accuracy, sensitivity, specificity, area under the receiver operating characteristic, and Nagelkerke’s R2 were compared. The ESMeta-analyses performed the worst, whereas the sum score and the ESGNB were worse than the ESLR that performed similar to the ESLASSO and ESRIDGE. The ESLR distinguished patients from controls (odds ratio [OR] = 1.94, P < .001), patients from siblings (OR = 1.58, P < .001), and siblings from controls (OR = 1.21, P = .001). An increase in ESLR was associated with a gradient increase of schizophrenia risk. In reference to the remaining fractions, the ESLR at top 30%, 20%, and 10% of the control distribution yielded ORs of 3.72, 3.74, and 4.77, respectively. Our findings demonstrate that predictive modeling approaches can be harnessed to evaluate the exposome.

 Expression of a Functional Recombinant Human Glycogen Debranching Enzyme (hGDE) in N. benthamiana Plants and in Hairy Root Cultures. Hernández MR¹, Triggiani D², Ivison F³, Demurtas OC⁴, Illiano E⁵, Marino C⁵, et al. BProtein Pept Lett. 2019 Oct 14. DOI: 10.2174/0929866526666191014154047. [Epub ahead of print]

Background Glycogen storage disease type III (GSDIII, Cori/Forbes disease) is a metabolic disorder due to the deficiency of the glycogen debranching enzyme (GDE), a large monomeric protein (about 176 kDa) with two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Several mutations along the Amylo-alpha-1,6-Glucosidase,4-Alpha-Glucanotransferase (Agl) gene are associated with loss of enzymatic activity. The unique treatment for GSDIII, at the moment, is based on diet. The potential of plants to manufacture exogenous engineered compounds for pharmaceutical purposes, from small to complex protein molecules such as vaccines, antibodies and other therapeutic/prophylactic entities, was shown by modern biotechnology through “Plant Molecular Farming”. Objective/Method In an attempt to develop novel protein-based therapeutics for GSDIII, the Agl gene, encoding for the human GDE (hGDE) was engineered for expression as a histidine-tagged GDE protein both in Nicotiana benthamiana plants by a transient expression approach, and in axenic hairy root in vitro cultures (HR) from Lycopersicum esculentum and Beta vulgaris. Results In both plant-based expression formats, the hGDE protein accumulated in the soluble fraction of extracts. The plant-derived protein was purified by affinity chromatography in native conditions showing glycogen debranching activity. Conclusion These investigations will be useful for the design of a new generation of biopharmaceuticals based on recombinant GDE protein that might represent, in the future, a possible therapeutic option for GSDIII.

Improving the sensitivity of an hsp20-based PCR for genus detection of Leishmania parasites in cutaneous clinical samples: a proof of concept. Montalvo AM, Alba A, Fraga J, Marzoa A, Torres C, Muskus C. Parasitol Res. 2019 Nov 27. DOI: 10.1007/s00436-019-06520-6. [Epub ahead of print]

Leishmaniasis is a parasitic disease of medical importance widely distributed around the world. Several methods are available for diagnosis but molecular approaches are highly recommended. To improve the sensitivity of an existing hsp20 gene based-PCR protocol to detect Leishmania parasites, primers were redesigned to amplify a shorter fragment using a new PCR variant (PCR-hsp20S). In this study, we aimed at characterizing the performance of the new method on cutaneous clinical samples and compare it with the former PCR-hsp20. The analytical sensitivity of the PCR-hsp20S was evaluated using DNA dilutions (100-0.1 pg) from Leishmania donovani and resulted in the detection of 10 fg of parasitic DNA, the equivalent to 0.05 parasite genome. For the diagnostic evaluation, a panel of 127 human clinical samples was used to calculate the parameters of sensitivity, specificity, accuracy, and positive and negative predictive values of the PCR-hsp20S. Diagnostic sensitivity was 94% (CI, 89.1-99.7%) and the specificity of 100% (CI, 98.6-100%). The same panel was also evaluated with the PCR-hsp20 to calculate the agreement between both molecular assays and to compare their performances. While both hsp20-based PCRs showed a good agreement coefficient (kappa index = 0.6), the performance of the novel variant, PCR-hsp20S, was significantly higher in terms of sensitivity (P = 0.0001) allowing the accurate detection of a higher number of Leishmania-positive clinical samples. We endorse the use of the PCR-hsp20S over the former protocol for the detection of Leishmania parasites from cutaneous clinical samples. In addition, as an improved sensitivity was achieved with the new method merely through the amplification of a shorter gene fragment, this investigation constitutes an experimental proof of this concept.

 Introduction of Application of Gini Coefficient to Heart Rate Variability Spectrum for Mental Stress Evaluation. Sánchez-Hechavarría ME, Ghiya S, Carrazana-Escalona R, Cortina-Reyna S, Andreu-Heredia A, Acosta-Batista C, et al. Arq Bras Cardiol. 2019 Sep 9. pii: S0066-782X2019005017103. DOI: 10.5935/abc.20190185. [Epub ahead of print]

Background The Gini coefficient is a statistical tool generally used by economists to quantify income inequality. However, it can be applied to any kind of data with unequal distribution, including heart rate variability (HRV). Objectives To assess the application of the Gini coefficient to measure inequality in power spectral density of RR intervals, and to use this application as a psychophysiological indicator of mental stress. Methods Thirteen healthy subjects (19 ± 1.5 years) participated in this study, and their RR intervals were obtained by electrocardiogram during rest (five minutes) and during mental stress (arithmetic challenge; five minutes). These RR intervals were used to obtain the estimates of power spectral densities (PSD). The limits for the PSD bands were defined from 0.15 to 0.40 Hz for high frequency band (HF), from 0.04 to 0.15 Hz for low frequency band (LF), from 0.04 to 0.085 Hz for first low frequency sub-band (LF1) and from 0.085 to 0.15 Hz for second low frequency sub-band (LF2). The spectral Gini coefficient (SpG) was proposed to measure the inequality in the power distribution of the RR intervals in each of above-mentioned HRV bands. SpG from each band was compared with its respective traditional index of HRV during the conditions of rest and mental stress. All the differences were considered statistically significant for p < 0.05. Results There was a significant decrease in HF power (p = 0.046), as well as significant increases in heart rate (p = 0.004), LF power (p = 0.033), LF2 power (p = 0.019) and LF/HF (p = 0.002) during mental stress. There was also a significant increase in SpG(LF) (p = 0.009) and SpG(LF2) (p = 0.033) during mental stress. Coefficient of variation showed SpG has more homogeneity compared to the traditional index of HRV during mental stress. Conclusions This pilot study suggested that spectral inequality of Heart Rate Variability analyzed using the Gini coefficient seems to be an independent and homogeneous psychophysiological indicator of mental stress. Also, HR, LF/HF, SpG(LF) of HRV are possibly important, reliable and valid indicators of mental stress.

Laryngeal Cancer Risks in Workers Exposed to Lung Carcinogens: Exposure-Effect Analyses Using a Quantitative Job Exposure Matrix. Hall AL, Kromhout H, Schüz J, Peters S, Portengen L, Vermeulen R, et al. Epidemiology. 2019 Oct 1.
DOI: 10.1097/EDE.0000000000001120. [Epub ahead of print]

Introduction Various established occupational lung carcinogens are also suspected risk factors for laryngeal cancer. However, individual studies are often inadequate in size to investigate this relatively rare outcome. Other limitations include imprecise exposure assessment and inadequate adjustment for confounders. Methods This study applied a quantitative job exposure matrix (SYN-JEM) for four established occupational lung carcinogens to five case-control studies within the INHANCE Consortium. We used occupational histories for 2256 laryngeal cancer cases and 7857 controls recruited from 1989-2007. We assigned quantitative exposure levels for asbestos, respirable crystalline silica, chromium-VI, and chromium-VI & nickel combined (to address highly correlated exposures) via SYN-JEM. We assessed effects of occupational exposure on cancer risk for males (asbestos, respirable crystalline silica, chromium-VI, chromium-VI & nickel) and females (asbestos, respirable crystalline silica), adjusting for age, study, tobacco smoking, alcohol consumption, and asbestos exposure where relevant. Results Among females, odds ratios (ORs) were increased for ever versus never exposed. Among males, p-values for linear trend were <0.05 for estimated cumulative exposure (all agents) and <0.05 for exposure duration (respirable crystalline silica, chromium-VI, and chromium-VI & nickel); strongest associations were for asbestos at >90%ile cumulative exposure (OR=1.3, CI=1.0-1.6), respirable crystalline silica at 30+ years duration (OR=1.4, CI=1.2-1.7) and 75%-90%ile cumulative exposure (OR=1.4, CI=1.1-1.8), chromium-VI at >75%ile cumulative exposure (OR=1.9, CI=1.2-3.0), and chromium-VI & nickel at 20-29 years duration (OR=1.5, CI=1.1-2.2). Conclusions These findings support hypotheses of causal links between four lung carcinogens (asbestos, respirable crystalline silica, chromium-VI, and nickel) and laryngeal cancer.

 Mangiferin: Possible uses in the prevention and treatment of mixed osteoarthritic pain. Garrido-Suárez BB, Garrido G, Piñeros O, Delgado-Hernández R. Phytother Res. 2019 Nov 22. DOI: 10.1002/ptr.6546. [Epub ahead of print]

Osteoarthritis (OA) pain has been proposed to be a mixed pain state, because in some patients, central nervous system factors are superimposed upon the more traditional peripheral factors. In addition, a considerable amount of preclinical and clinical evidence has shown that, accompanying the central neuroplasticity changes and partially driven by a peripheral nociceptive input, a real neuropathic component occurs that are particularly linked to disease severity and progression. Hence, innovative strategies targeting neuroprotection and particularly neuroinflammation to prevent and treat OA pain could be introduced. Mangiferin (MG) is a glucosylxanthone that is broadly distributed in higher plants, such as Mangifera indica L. Previous studies have documented its analgesic, anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory properties. In this paper, we propose its potential utility as a multitargeted compound for mixed OA pain, even in the context of multimodal pharmacotherapy. This hypothesis is supported by three main aspects: the cumulus of preclinical evidence around this xanthone, some preliminary clinical results using formulations containing MG in clinical musculoskeletal or neuropathic pain, and by speculations regarding its possible mechanism of action according to recent advances in OA pain knowledge.

 Modulation of CD4 T cell function via CD6-targeting. Freitas RF, Basto A, Almeida SCP, Santos RF, Gonçalves CM, Corria-Osorio J, et al. EBioMedicine. 2019 Sep;47:427–35.

In recent years molecules involved on the immune synapse became successful targets for therapeutic immune modulation. CD6 has been extensively studied, yet, results regarding CD6 biology have been controversial, in spite of the ubiquitous presence of this molecule on virtually all CD4 T cells. We investigated the outcome of murine and human antibodies targeting CD6 domain 1. We found that CD6-targeting had a major impact on the functional specialization of CD4 cells, both human and murine. Differentiation of CD4 T cells towards a Foxp3⁺ Treg fate was prevented with increasing doses of anti-CD6, while Th1 polarization was favoured. No impact was observed on Th2 or Th17 specialization. These in vitro results provided an explanation for the dose-dependent outcome of in vivo anti-CD6 administration where the anti-inflammatory action is lost at the highest doses. Our data show that therapeutic targeting of the immune synapse may lead to paradoxical dose-dependent effects due to modification of T cell fate.

 Molecular docking and in vitro evaluation of a new hybrid molecule (JM-20) on cholinesterase activity from different sources. D’Avila da Silva F, Nogara PA, Ochoa-Rodríguez E, Núñez-Figueredo Y, Wong-Guerra M, Rosemberg DB, et al. Biochimie. 2019 Nov 23. pii: S0300-9084(19)30334-7. DOI: 10.1016/j.biochi.2019.11.011. [Epub ahead of print]

The main function of AChE is the hydrolysis of the neurotransmitter acetylcholine (ACh) at the neuromuscular and in cholinergic brain synapses. In some pathologies, loss of cholinergic neurons may be associated with a deficiency of ACh in specific brain areas. Consequently, the study of new safe drugs that inhibit AChE is important, because they can increase ACh levels in the synaptic cleft without adverse effects. Here, we evaluated the effects of JM-20 (a benzodiazepine-dihydropyridine hybrid molecule) on cholinesterase (ChE) activities from distinct sources (AChE from Electrophorus electricus (EeAChE), human erythrocyte membranes (HsAChE (ghost)), total erythrocyte (HsAChE (erythrocyte)) and BChE from plasma (HsBChE) and purified enzyme from the horse (EcBChE)). Kinetic parameters were determined in the presence of 0.05-1.6 mM of substrate concentration. The interactions ChEs with JM-20 were performed using molecular docking simulations. JM-20 inhibited all tested AChE but not BChE. The IC₅₀ values were 123.0 nM ± 0.18 (EeAChE), 158.1 nM ± 0.14 (ghost HsAChE), and 172.4 nM ± 0.16 (erythrocytic HsAChE). JM-20 caused a mixed type of inhibition (it altered K_m and V_max of AChE). The molecular docking indicated the binding poses and the most plausible active isomer of JM-20. Besides giving important data for future drug design, our results help us understand the mode of action of JM-20 as a specific inhibitor of AChE enzymes.

National survey of pre-treatment HIV drug resistance in Cuban patients. Machado LY, Blanco M, López LS, Díaz HM, Dubed M, Valdés N, et al. PLoS One. 2019 Sep 3;14(9):e0221879. DOI: 10.1371/journal.pone.0221879.

Background The World Health Organization (WHO) recommends a method to estimate nationally representative pretreatment HIV drug resistance (PDR) in order to evaluate the effectiveness of first -line treatments. The objective of the present study was to determine the prevalence of PDR in Cuban adults infected with HIV-1. Materials and methods A cross-sectional study in Cuban adults infected with HIV-1 over 18 years was conducted. The probability proportional to size method for the selection of municipalities and patients without a prior history of antiretroviral treatment during the period from January 2017 to June 2017 was used. The plasma from 141 patients from 15 municipalities for the determination of viral subtype and HIV drug resistance was collected. Some clinical and epidemiological variables were evaluated. Results 80. 9% of the patients corresponded to the male sex and 76.3% were men who have sex with other men (MSM). The median CD4 count was 371 cells / mm3 and the median viral load was 68000 copies / mL. The predominant genetic variants were subtype B (26.9%), CRF19_cpx (24.1%), CRF 20, 23, 24_BG (23.4%) and CRF18_cpx (12%). Overall, the prevalence of PDR was 29.8% (95%, CI 22.3-38.1). The prevalence was 12.8% (95%, CI 6.07-16.9) for any nucleoside reverse transcriptase inhibitor (NRTI), 23.4% (95%, CI 16.7-31.3) for any non-reverse transcriptase inhibitor (NNRTI) and 1.4% (95%, CI 0.17-5.03) for any protease inhibitor (PI). The most frequent mutations detected were K103N (12.9%), G190A (6.4%) and Y181C (4.8%). Conclusions The NNRTI prevalence above 10% in our study indicates that the first-line antiretroviral therapy in Cuba may be less effective and supports the need to look for new treatment options that contribute to therapeutic success and help the country achieve the global goals 90-90-90 set forth by UNAIDS.

 Neuroinflammation and Neuromodulation in Neurological Diseases. Robinson-Agramonte MLA, Gonçalves CA, Farina de Almeida R, González Quevedo A, Chow S, Velázquez Pérez L, et al. Behav Sci (Basel). 2019 Sep 12;9(9). pii: E99. DOI: 10.3390/bs9090099.

Neuroimmunology is a relatively young science. This discipline has emerged today from the research field as a mature and fully developed innovative research area that integrates not only pure topics of neuroimmunology, but also expands on wider fields such as neuroplasticity, neuronal reserve and neuromodulation in association with clinical events, amongst which behavioral disorders stand out. The Cuban School of Neuroimmunology-a recent meeting that took place in Havana, Cuba-focused on topics based on the molecular mechanisms of neuroinflammation in neurological disorders involving behavioral manifestations, such as multiple sclerosis (MS), autism, cerebellar ataxias, Alzheimer´s disease and stroke among others, as well as on the use of new interventional technologies in neurology. Professor Luis Velazquez, from the Cuban Academy of Sciences, dictated an interesting lecture on Spinocerebellar ataxias, a genetic disorder where recent hypotheses related to the influence of neuroinflammation as a neurobiological factor influencing the progression of this disease have emerged. At the same time, the use of new interventional technologies in neurology was discussed, including those referring to novel disease modifying therapies in the course of MS and the use of transcranial magnetic stimulation in several neurological diseases, the latter reinforcing how interventional strategies in the form of non-invasive bran stimulation can contribute to physical rehabilitation in neurology. This paper summarizes the highlights of the most relevant topics presented during the First Cuban School of Neuroimmunology, organized by the Cuban Network of Neuroimmunology, held in June 2019.

Nurr1, Pitx3, and α7 nAChRs mRNA Expression in Nigral Tissue of Rats with Pedunculopontine Neurotoxic Lesion. Blanco-Lezcano L, Alberti-Amador E, González-Fraguela ME, Larrea GZ, Pérez-Serrano RM, Jiménez-Luna NA, et al. Medicina (Kaunas). 2019 Sep 20;55(10). pii: E616. doi: 10.3390/medicina55100616.

Background and objectives The knowledge that the cholinergic neurons from pedunculopontine nucleus (PPN) are vulnerable to the degeneration in early stages of the Parkinson disease progression has opened new perspectives to the development of experimental model focused in pontine lesions that could increase the risk of nigral degeneration. In this context it is known that PPN lesioned rats exhibit early changes in the gene expression of proteins responsible for dopaminergic homeostasis. At the same time, it is known that nicotinic cholinergic receptors (nAChRs) mediate the excitatory influence of pontine-nigral projection. However, the effect of PPN injury on the expression of transcription factors that modulate dopaminergic neurotransmission in the adult brain as well as the α7 nAChRs gene expression has not been studied. The main objective of the present work was the study of the effects of the unilateral neurotoxic lesion of PPN in nuclear receptor-related factor 1 (Nurr1), paired-like homeodomain transcription factor 3 (Pitx3), and α7 nAChRs mRNA expression in nigral tissue. Materials and Methods: The molecular biology studies were performed by means of RT-PCR. The following experimental groups were organized: Non-treated rats, N-methyl-D-aspartate (NMDA)-lesioned rats, and Sham operated rats. Experimental subjects were sacrificed 24 h, 48 h and seven days after PPN lesion. Results Nurr1 mRNA expression, showed a significant increase both 24 h (p < 0.001) and 48 h (p < 0.01) after PPN injury. Pitx3 mRNA expression evidenced a significant increase 24 h (p < 0.001) followed by a significant decrease 48 h and seven days after PPN lesion (p < 0.01). Finally, the α7 nAChRs nigral mRNA expression remained significantly diminished 24 h, 48 h (p < 0.001), and 7 days (p < 0.01) after PPN neurotoxic injury. Conclusion Taking together these modifications could represent early warning signals and could be the preamble to nigral neurodegeneration events.

New formulation of the Gompertz equation to describe the kinetics of untreated tumors. Castañeda ARS, Torres ER, Goris NAV, González MM, Reyes JB, González VGS, et al  PLoS One. 2019 Nov 12;14(11):e0224978. DOI: 10.1371/journal.pone.0224978.

Background Different equations have been used to describe and understand the growth kinetics of undisturbed malignant solid tumors. The aim of this paper is to propose a new formulation of the Gompertz equation in terms of different parameters of a malignant tumor: the intrinsic growth rate, the deceleration factor, the apoptosis rate, the number of cells corresponding to the tumor latency time, and the fractal dimensions of the tumor and its contour. Methods Furthermore, different formulations of the Gompertz equation are used to fit experimental data of the Ehrlich and fibrosarcoma Sa-37 tumors that grow in male BALB/c/Cenp mice. The parameters of each equation are obtained from these fittings. Results The new formulation of the Gompertz equation reveals that the initial number of cancerous cells in the conventional Gompertz equation is not a constant but a variable that depends nonlinearly on time and the tumor deceleration factor. In turn, this deceleration factor depends on the apoptosis rate of tumor cells and the fractal dimensions of the tumor and its irregular contour. Conclusions It is concluded that this new formulation has two parameters that are directly estimated from the experiment, describes well the growth kinetics of unperturbed Ehrlich and fibrosarcoma Sa-37 tumors, and confirms the fractal origin of the Gompertz formulation and the fractal property of tumors.

OSU-6162, a Sigma1R Ligand in Low Doses, Can Further Increase the Effects of Cocaine Self-Administration on Accumbal D2R Heteroreceptor Complexes. Borroto-Escuela DO, Romero-Fernández W, Wydra K, Zhou Z, Suder A, Filip M, et al. Neurotox Res. 2019 Nov 28. DOI: 10.1007/s12640-019-00134-7. [Epub ahead of print]

Cocaine was previously shown to act at the Sigma1R which is a target for counteracting cocaine actions. It therefore becomes of interest to test if the monoamine stabilizer (-) OSU-6162 (OSU-6162) with a nanomolar affinity for the Sigma1R can acutely modulate in low doses the effects of cocaine self-administration. In behavioral studies, OSU-6162 (5 mg/kg, s.c.) did not significantly change the number of active lever pressing and cocaine infusions. However, a trend to reduce cocaine readouts was found after 3 days of treatment. In contrast, in maintenance of cocaine self-administration, the proximity ligation assay performed on brains from rats pretreated with OSU-6162 showed highly significant increases in the density of the D2R-Sigma1R heteroreceptor complexes in the shell of the nucleus accumbens versus OSU-6162 induced increases in this region of yoked saline rats. In cocaine self-administration, highly significant increases were also induced by OSU-6162 in the A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell versus vehicle-treated rats. Furthermore, ex vivo, the A2AR agonist CGS21680 (100 nM) produced a marked and significant increase of the D2R Ki high values in the OSU-6162-treated versus vehicle-treated rats under maintenance of cocaine self-administration. These results indicate a substantial increase in the inhibitory allosteric A2AR-D2R interactions following cocaine self-administration upon activation by the A2AR agonist ex vivo. The current results indicate that OSU-6162 via its high affinity for the Sigma1R may increase the number of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes associated with further increases in the antagonistic A2AR-D2R interactions in cocaine self-administration.

Phycocyanobilin reduces brain injury after endothelin-1- induced focal cerebral ischemia. Pavón-Fuentes N, Marín-Prida J, Llópiz-Arzuaga A, Falcón-Cama V, Campos-Mojena R, Cervantes-Llanos M, et al. Clin Exp Pharmacol Physiol. 2019 Nov 15.
DOI: 10.1111/1440-1681.13214. [Epub ahead of print]

Pharmacological therapies for interrupting biochemical events of the ischemic cascade and protecting against stroke in humans are as yet unavailable. Up to now, the neuroprotective activity in cerebral ischemia of Phycocyanobilin (PCB), a tetrapyrrolic natural antioxidant, has not been fully examined. Here, we evaluated if PCB protects PC12 neuronal cells against oxygen and glucose deprivation plus reperfusion, and its protective effects in a rat model of endothelin-1-induced focal brain ischemia. PCB was purified from the cyanobacteria Spirulina platensis and characterized by spectrophotometric, liquid and gas chromatography and mass spectrometry techniques. In Wistar rats, PCB at 50, 100 and 200 µg/kg or phosphate-buffered saline (vehicle) were administered intraperitoneally at equal subdoses in a therapeutic schedule (30 minutes, 1, 3 and 6 h after the surgery). Brain expression of myelin basic protein (MBP) and the enzyme CNPase was determined by immunoelectron microscopy. PCB was obtained with high purity (>95%) and the absence of solvent contaminants, and was able to ameliorate PC12 cell ischemic injury. PCB treatment significantly decreased brain infarct volume, limited the exploratory behavior impairment and preserved viable cortical neurons in ischemic rats in a dose-dependent manner, compared to the vehicle group. Furthermore, PCB at high doses restored the MBP and CNPase expression levels in ischemic rats. An improved PCB purification method from its natural source is reported, obtaining PCB that is suitable for pharmacological trials showing neuroprotective effects against experimental ischemic stroke. Therefore, PCB could be a therapeutic pharmacological alternative for ischemic stroke patients.

 Prevalence and incidence of chronic kidney disease in Cuba. Herrera Valdés R, Almaguer López M, Chipi Cabrera JA, Pérez-Oliva Díaz JF, Landrove Rodríguez O, Mármol Sóñora A. Clin Nephrol. 2019 Sep 24. DOI: 10.5414/CNP92S111. [Epub ahead of print]

Chronic kidney disease (CKD) is a health problem worldwide. This article’s objective is to describe CKD’s integration into Cuba’s National Noncommunicable Diseases (NCD) Program and the main outcomes regarding the burden of CKD and associated risk factors in Cuba. Cuba offers free health services to all its citizens on the basis of a strong primary healthcare system focused on prevention. The CKD National Program is coordinated by the Institute of Nephrology and includes the National Program for Prevention of CKD, which addresses all levels of prevention. The following indicators for renal replacement treatment are from 2016. The incidence of new patients on dialysis was 109 per million population (pmp); the two main causes were hypertension (34.4%) and diabetes mellitus (29.2). In 6.3% of patients, CKD cause could not be determined because they presented at advanced stages. The prevalence of patients on dialysis was 289 pmp; 90% of dialysis patients were on hemodialysis. The main causes of death were cardiovascular diseases (30.25%), cerebrovascular diseases (11.1%), and infections (29.5%). The kidney transplant rate was 14.3 pmp. Kidney transplants performed with cadaveric donors were 86.5% of total, with living related donors 13.5%. The Isle of Youth Study (ISYS) was designed to assess predialysis chronic kidney disease patterns; its methodology has been published previously. Results: Risk factors: age > 59 years 32%, women 67.8%, overweight 34.3%, obesity 22.8%, hypertension 41.5%, diabetes 13%. Estimated CKD prevalence was 9.63%. The integration of CKD into Cuba’s NCD Program has gathered knowledge.

 Rapanone, a naturally occurring benzoquinone, inhibits mitochondrial respiration and induces HepG2 cell death. Pardo Andreu GL, Reis FZD, González-Durruthy M, Hernández RD, D’Vries RF, Vanden Berghe W, et al. Toxicol In Vitro. 2019 Nov 20;63:104737.
DOI: 10.1016/j.tiv.2019.104737. [Epub ahead of print]

 Rapanone is a natural occurring benzoquinone with several biological effects including unclear cytotoxic mechanisms. Here we addressed if mitochondria are involved in the cytotoxicity of rapanone towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In the HepG2, rapanone (20-40 μM) induced a concentration-dependent mitochondrial membrane potential dissipation, ATP depletion, hydrogen peroxide generation and, phosphatidyl serine externalization; the latter being indicative of apoptosis induction. Rapanone toxicity towards primary rats hepatocytes (IC₅₀ = 35.58 ± 1.50 μM) was lower than that found for HepG2 cells (IC₅₀ = 27.89 ± 0.75 μM). Loading of isolated mitochondria with rapanone (5-20 μM) caused a concentration-dependent inhibition of phosphorylating and uncoupled respirations supported by complex I (glutamate and malate) or the complex II (succinate) substrates, being the latter eliminated by complex IV substrate (TMPD/ascorbate). Rapanone also dissipated mitochondrial membrane potential, depleted ATP content, released Ca²⁺ from Ca²⁺-loaded mitochondria, increased ROS generation, cytochrome c release and membrane fluidity. Further analysis demonstrated that rapanone prevented the cytochrome c reduction in the presence of decylbenzilquinol, identifying complex III as the site of its inhibitory action. Computational docking results of rapanone to cytochrome bc1 (Cyt bc1) complex from the human sources found spontaneous thermodynamic processes for the quinone-Q_o and Q_i binding interactions, supporting the experimental in vitro assays. Collectively, these observations suggest that rapanone impairs mitochondrial respiration by inhibiting electron transport chain at Complex III and promotes mitochondrial dysfunction. This property is potentially involved in rapanone toxicity on cancer cells.

 Repeat-Dose Toxicity Study Using the AFPL1-Conjugate Nicotine Vaccine in Male Sprague Dawley Rats. Oliva R, Fraleigh NL, Lewicky JD, Fariñas M, Hernández T, Martel AL, et al. Pharmaceutics. 2019 Nov 23;11(12). pii: E626.
DOI: 10.3390/pharmaceutics11120626.

Tobacco smoking is the cause of 20% of Canadian deaths per year. Nicotine vaccines present a promising alternative to traditional smoking cessation products, but to date, no vaccine has been able to move through all phases of clinical trials. We have previously demonstrated that the AFPL1-conjugate nicotine vaccine does not induce systemic or immunotoxicity in a mouse model and that a heterologous vaccination approach is more advantageous than the homologous routes to inducing mucosal and systemic anti-nicotine antibodies. The purpose of this study was to confirm the safety profile of the vaccine in a repeat-dose toxicity study. The heterologous vaccination strategy was again used, and Sprague Dawley rats were administered a dose five times greater than in our previous studies. Physiological conditions, food and water consumption, body temperature, injection site inflammation, relative weights of organs, histopathology, and blood chemistry and hematology were evaluated during the course of the vaccination period to determine the safety of the vaccine. The AFPL1-conjugate nicotine vaccine did not induce clinically relevant changes or induce symptoms that would be associated with toxicity, making it a promising candidate for future investigations.

 Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation. Ramos Y, González A, Sosa-Acosta P, Pérez-Riverol Y, García Y, Castellanos-Serra L, et al. J Sep Sci. 2019 Oct 17. DOI: 10.1002/jssc.201900495. [Epub ahead of print]

Shotgun proteomics based on peptide fractionation via liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation via electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis for protein fractionation and Sodium Dodecyl Sulfate free Polyacrylamide Gel Electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for Sodium Dodecyl Sulfate free Polyacrylamide Gel Electrophoresis improving peptide recoveries respect to the established electrophoretic technique Off Gel Electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device allows simultaneously the separation and the collection of fractionated peptides in solution.

 The BioDoseNet image repository used as a training tool for the dicentric assay. García O, Rada-Tarifa A, Lafuente-Álvarez E, González-Mesa JE, Mandina T, Muñoz-Velastegui G, et al. Int J Radiat Biol. 2019 Sep 20:1–9. DOI: 10.1080/09553002.2019.1665211. [Epub ahead of print]

Purpose This paper describes how the BioDoseNet image repository was used as a training tool for the dicentric assay. Materials and methods The training was implemented in three phases: introduction to dicentric scoring, dose response curve elaboration and dose assessment exercise. Four labs without previous experience in the dicentric assay participated and four modules of the repository were used. Results The labs become familiar with aberrations induced by ionizing radiation. The labs were able to generate data for the elaboration of a dose response curve and then successfully estimated doses and irradiated fractions in six blind samples. Conclusions The performance of these laboratories during the exercise demonstrates the efficacy of the BioDoseNet image repository as a training tool and the utility of web based scoring for the dicentric assay community.

Undersampling: case studies of flaviviral inhibitory activities. Barigye SJ, García de la Vega JM, Castillo-Garit JA. J Comput Aided Mol Des. 2019 Nov 26.
DOI: 10.1007/s10822-019-00255-3. [Epub ahead of print]

Imbalanced datasets, comprising of more inactive compounds relative to the active ones, are a common challenge in ligand-based model building workflows for drug discovery. This is particularly true for neglected tropical diseases since efforts to identify therapeutics for these diseases are often limited. In this report, we analyze the performance of several undersampling strategies in modeling the Dengue Virus 2 (DENV2) inhibitory activity, as well as the anti-flaviviral activities for the West Nile (WNV) and Zika (ZIKV) viruses. To this end, we build datasets comprising of 1218 (159 actives and 1059 inactives), 1044 (132 actives and 912 inactives) and 302 (75 actives and 227 inactives) molecules with known DENV2, WNV and ZIKV inhibitory activity profiles, respectively. We develop ensemble classifiers for these endpoints and compare the performance of the different undersampling algorithms on external sets. It is observed that data pruning algorithms yield superior performance relative to data selection algorithms. The best overall performance is provided by the one-sided selection algorithm with test set balanced accuracy (BACC) values of 0.84, 0.74 and 0.77 for the DENV2, WNV and ZIKV inhibitory activities, respectively. For the model building, we use the recently proposed GT-STAF information indices, and compare the predictivity of 3 molecular fragmentation approaches: connected subgraphs, substructure and alogp atom types, which are observed to show comparable performance. On the other hand, a combination of indices based on these fragmentation strategies enhances the predictivity of the built ensembles. The built models could be useful for screening new molecules with possible DENV, WNV and ZIKV inhibitory activities. ADMET modelers are encouraged to adopt undersampling algorithms in their workflows when dealing with imbalanced datasets.

 ZikaPLAN: addressing the knowledge gaps and working towards a research preparedness network in the Americas. Wilder-Smith A, Preet R, Brickley EB, Ximenes RAA, Miranda-Filho DB, Turchi Martelli CM, et al. Glob Health Action. 2019;12(1):1666566.
DOI: 10.1080/16549716.2019.1666566.

Zika Preparedness Latin American Network (ZikaPLAN) is a research consortium funded by the European Commission to address the research gaps in combating Zika and to establish a sustainable network with research capacity building in the Americas. Here we present a report on ZikaPLAN`s mid-term achievements since its initiation in October 2016 to June 2019, illustrating the research objectives of the 15 work packages ranging from virology, diagnostics, entomology and vector control, modelling to clinical cohort studies in pregnant women and neonates, as well as studies on the neurological complications of Zika infections in adolescents and adults. For example, the Neuroviruses Emerging in the Americas Study (NEAS) has set up more than 10 clinical sites in Colombia. Through the Butantan Phase 3 dengue vaccine trial, we have access to samples of 17,000 subjects in 14 different geographic locations in Brazil. To address the lack of access to clinical samples for diagnostic evaluation, ZikaPLAN set up a network of quality sites with access to well-characterized clinical specimens and capacity for independent evaluations. The International Committee for Congenital Anomaly Surveillance Tools was formed with global representation from regional networks conducting birth defects surveillance. We have collated a comprehensive inventory of resources and tools for birth defects surveillance, and developed an App for low resource regions facilitating the coding and description of all major externally visible congenital anomalies including congenital Zika syndrome. Research Capacity Network (REDe) is a shared and open resource centre where researchers and health workers can access tools, resources and support, enabling better and more research in the region. Addressing the gap in research capacity in LMICs is pivotal in ensuring broad-based systems to be prepared for the next outbreak. Our shared and open research space through REDe will be used to maximize the transfer of research into practice by summarizing the research output and by hosting the tools, resources, guidance and recommendations generated by these studies. Leveraging on the research from this consortium, we are working towards a research preparedness network.